Compositions and methods for differential regulation of fatty acid unsaturation in membrane lipids and seed oil

ABSTRACT

Aspects of the invention provide methods for differential regulation of fatty acid unsaturation in seed oil and membrane lipids of plants based on modulation of a previously unknown biosynthetic pathway involving a novel phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) that regulates phosphatidylcholine biosynthesis in developing oil seed plants. Specific aspects relate to inventive PDCT polypeptides including, for example, variants, deletions, muteins, fusion proteins, and orthologs thereof (collectively PDCT proteins), to nucleic acids encoding same, to plants comprising such PDCT sequences or proteins or devoid or depleted of such PDCT proteins or sequences, and to methods for generating plants having altered or no PDCT expression and/or activity, including but not limited to methods comprising mutagenesis, recombinant DNA, transgenics, etc.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/921,119, filed Feb. 1, 2011, which is the United States nationalstage, pursuant to 35 U.S.C. § 371, of International Patent ApplicationNo. PCT/US2009/036066, filed Mar. 4, 2009, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 61/149,288, filedFeb. 2, 2009, and U.S. Provisional Patent Application No. 61/033,742,filed Mar. 4, 2008, all of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made, at least in part, with Government support undergrants 2006-35318-17797 and 97-35301-4426 awarded by the United StatesDepartment of Agriculture (USDA), and the United States Government,therefore, has certain rights in the invention.

SEQUENCE LISTING

A Sequence Listing comprising SEQ ID NOS:1-30, has been provided incomputer readable form (.txt) as part of this application, and isincorporated by reference herein in its entirety as part of thisapplication.

FIELD OF THE INVENTION

Aspects of the invention relate generally to fatty acid biosynthesis,membrane lipids and plant seed oils, and more particularly tobiosynthesis of unsaturated fatty acids and related acylglycerols and tocompositions and methods for differential regulation of fatty acidunsaturation in seed oil and membrane lipids of plants based onmodulation of a previously unknown biosynthetic pathway involving anovel phosphatidylcholine:diacylglycerol cholinephosphotransferase(PDCT) that regulates phosphatidylcholine biosynthesis in developingoilseed plants (e.g., of Arabidopsis, soybean (Glycine max), canola(Brassica napus or B. rapa), sunflower (Helianthus annuus), etc.).Specific aspects relate to inventive PDCT polypeptides including, forexample, variants, deletions, muteins, fusion proteins, and orthologsthereof (collectively PDCT proteins), to isolated nucleic acids encodingsame, to plants comprising such PDCT proteins or devoid of such PDCTproteins, and to methods for generating plants having altered or no PDCTexpression and/or activity, including but not limited to methodscomprising mutagenesis, gene-silencing, antisense, siRNA, recombinantDNA, transgenics, etc.).

BACKGROUND

Many plant species including, for example, Arabidopsis thaliana storetriacylglycerols (TAGs) in their seeds as a carbon reserve. These TAGsare the major source of energy and carbon material that supportsseedling development during the early stages of plant life. Vegetableoils from soybean (Glycine max), canola (Brassica napus or B. rapa),sunflower (Helianthus annuus) and many other oilseed crops are also animportant source of oil for the human diet or industrial applicationsincluding, but not limited to biofuels, biolubricants, nylon precursors,and detergent feedstocks. The degree and/or amount of polyunsaturatedfatty acids of vegetable oils are characteristic and determinativeproperties with respect to oil uses in food or non-food industries. Morespecifically, the characteristic properties and utilities of vegetableoils are largely determined by their fatty acyl compositions in TAG.

Major vegetable oils are comprised primarily of palmitic (16:0), stearic(18:0), oleic (18:1cis Δ⁹), linoleic (18:2cis Δ^(9, 12)), andα-linolenic (18:3cis Δ9, 12, 15) acids. Modifications of the fatty acidcompositions have been sought after for at least a century in order toprovide optimal oil products for human nutrition and chemical (e.g.,oleochemical) uses (1-3). In particular, the polyunsaturated fatty acids(18:2 and 18:3) have received considerable attention because they aremajor factors that affect nutritional value and oil stability. However,while these two fatty acids provide essential nutrients for humans andanimals, they increase oil instability because they comprise multipledouble bonds that may be easily oxidized during processing and storage.

Limitations of the Art.

The desaturation of 18:1 into 18:2 is a critical step for synthesizingpolyunsaturated fatty acids. During storage lipid biosynthesis, thisreaction is known to be catalyzed by the fatty acid desaturase, FAD2, amembrane-bound enzyme located on the endoplasmic reticulum (ER) (4). TheFAD2 substrate 18:1 must be esterified on the sn-2 position ofphosphatidylcholine (PC) (5, 6), which is the major membranephospholipid of plant cells. Not surprisingly, therefore,down-regulation of FAD2 (and FAD3) genes has become a preferred strategyfor avoiding the need to hydrogenate vegetable oils and the concomitantproduction of undesirable trans fatty acids. For example, soybean hasboth seed-specific and constitutive FAD2 desaturases, so that genesilencing of the seed-specific isoform has allowed the production ofhigh-oleate cultivars (>88% 18:1 in the oil) in which membraneunsaturation and plant performance are largely unaffected.Significantly, however, such FAD2 gene-silencing strategies aresubstantially limited because, for example, canola and other oilseedplants have only constitutive FAD2 enzymes. Therefore, in canola andother such constitutive FAD2 crops, silencing or down-regulation of FAD2not only alters the fatty acid composition of the storagetriacylglycerol (TAG) in seeds, but also of the cellular membranes,which severely compromises growth and yield of the plant. For example,the defective FAD2 in the Arabidopsis mutant fad2 alters fatty acidcompositions of seeds as well as vegetable tissues, and severelycompromises plant growth (4). FAD2 mutations and silencing that producethe highest 18:1 levels in the oil also reduce membrane unsaturation invegetative and seed tissues, resulting in plants that germinate and growpoorly. As a result, only partial downregulation of FAD2 expression ispossible, producing approximately 70-75% 18:1 in the oil of commercialcultivars such as Nexera/Natreon (Dow AgroSciences) and Clear Valley 75(Cargill).

There is, therefore, a pronounced need in the art for novel compositionsand methods for differential regulation of fatty acid unsaturation inseed oil and membrane lipids of plants, and for viable plants (e.g.,canola, etc.) having reduced fatty acid unsaturation in seed oils,without deleterious alterations in the unsaturation of membrane lipidcomponents.

SUMMARY OF EXEMPLARY EMBODIMENTS

Particular aspects provide novel compositions and methods fordifferential regulation of fatty acid unsaturation in seed oil andmembrane lipids, without deleterious alterations in the unsaturation ofmembrane lipid components.

Additional aspects provide compositions and methods for differentialregulation of fatty acid unsaturation in seed oil and membrane lipids ofplants, based on modulation of a previously unknown biosynthetic pathwayinvolving a novel phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) that regulates phosphatidylcholinebiosynthesis in developing oilseed plants (e.g., of Arabidopsis, soybean(Glycine max), canola (Brassica napus or B. rapa), sunflower (Helianthusannuus), etc.).

Further aspects provide inventive PDCT polypeptides including, forexample, variants, deletions, muteins, fusion proteins, and orthologsthereof (collectively PDCT proteins).

Yet additional aspects provide plants comprising such PDCT sequences orproteins or devoid or depleted of such PDCT proteins or sequences, andmethods for generating plants having altered or no PDCT expressionand/or activity, including but not limited to methods comprisingmutagenesis, gene-silencing, antisense, siRNA, recombinant DNA,transgenics, etc.).

Specific aspects provide a method for regulation of fatty acidunsaturation in seed oil, comprising: obtaining an oilseed-bearingplant; and modulating the expression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inone or more seeds or developing seeds of the plant, wherein the level,amount, or distribution of fatty acid unsaturation in the seed oil ismodified relative to the seed oil of plants with normal seed expressionof the PDCT. In certain embodiments, modulating the expression oractivity of the at least one PDCT comprises down-regulating theexpression or activity, wherein the level, amount, or distribution offatty acid unsaturation in the seed oil is modified is reduced.Preferably, the method comprises differential regulation of fatty acidunsaturation in seed oil relative to fatty acid unsaturation in one ormore membrane lipids. Preferably, the fatty acid unsaturation in seedoil relative to fatty acid unsaturation in one or more membrane lipidsis differentially reduced in seed oil.

Additional aspects provide a method of producing an oil seed-bearingplant or a part thereof, comprising imparting into the germplasm of anoil seed-bearing plant variety a mutation or genetic alteration thatmodifies the expression or activity of at least one PDCT in one or moreseeds or developing seeds of the plant, wherein the level, amount, ordistribution of fatty acid unsaturation in the seed oil is modifiedrelative to the seed oil of plants with normal seed expression of thePDCT.

Further embodiments comprise an oil seed-bearing plant or a partthereof, comprising a mutation or genetic alteration that modifies theexpression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inone or more seeds or developing seeds of the plant, wherein the level,amount, or distribution of fatty acid unsaturation in the seed oil ismodified relative to the seed oil of plants with normal seed expressionof the PDCT. While the mutation or genetic alteration may be one thatmodulates PDCT expression and/or activity directly or indirectly, inparticular aspects the mutation comprises a mutation of at least onePDCT sequence that modifies the expression or activity thereof in one ormore seeds or developing seeds of the plant, wherein the level, amount,or distribution of fatty acid unsaturation in the seed oil is modifiedrelative to the seed oil of plants with normal seed expression of thePDCT.

Additional aspects provide a seed or true-breeding seed derived from theoil seed-bearing plants or parts thereof as provided for herein.

Further aspects provide an oil derived from the oil seed-bearing plantsor parts thereof as provided for herein.

Yet additional embodiments provide a fuel (e.g., bio-fuel), based atleast in part on at least one oil derived from the oil seed-bearingplants or parts thereof as provided for herein.

BRIEF SUMMARY OF THE DRAWINGS

FIGS. 1A-1D show lipid synthesis in developing seeds of Arabidopsis.Developing seeds were labeled with radioactive acetate (to label fattyacids) (FIGS. 1C and 1D) and radioactive glycerol (to label the lipidbackbone) (FIGS. 1A and 1B). After 15 min of pulse with [14-C] labeledglycerol (C) or acetate (D), the chase was carried out for 180 min.Radio activity in PC, DG and TG were determined at 0, 30, 60 and 180 mintime points;

FIGS. 1E-1H show a comparison of fatty acid composition between rod1 andWT in TG, DG, PC and PE from developing seeds at 9 days after flowering.

FIGS. 2A-2C show that the ROD1 gene was identified as At3g15820 inArabidopsis;

FIG. 2D shows map-based identification of the ROD1 Locus on Arabidopsischromosome 3;

FIGS. 3A-3C show that the ROD1 functions as aphosphatidylcholine:diacylglycerol cholinephosphotransferase;

FIG. 4 shows the ROD1 mutant truncated amino acid sequence (SEQ ID NO:5)in DH4. According to particular aspects, aphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)mutant (rod1) coding sequence (SEQ ID NO:4) comprises a G>A change atnucleotide position 228, resulting in premature termination of the PDCTprotein to provide a 75 amino acid truncated ROD1 mutant sequence (SEQID NO:5);

FIG. 5 shows, according to particular exemplary aspects, primarysequence relationships between LPT family members. According toparticular aspects, ROD1 belongs to a lipidphosphatase/phosphotransferase family;

FIG. 6 shows, according to particular exemplary aspects, known sequenceshomologous to the Arabidopsis ROD1 in different organisms. According toparticular aspects, ROD1 regulates Equilibration between Diacylglyceroland Phosphatidylcholine in Oilseeds;

FIG. 7 shows, according to particular exemplary aspects, RT-PCR of ROD1and At3g15830 expression in Arabidopsis and yeast cells. Lanes 1-8 areresults for ROD1, and lanes 9-16 are for At3g15830. RT-PCR samples aretotal RNA from germinating seedlings (1,9), young leaves (2, 10),flowers (3,11), siliques (4, 12) of WT Arabidopsis and siliques fromrod1 mutant plants (5, 13); yeast cells containing p424GPD (7, 15) orp424ROD1 (8) and p424-At3g15830 (16); and genomic DNA from rod1 (6, 14);

FIG. 8 shows, according to particular exemplary aspects, digitalNorthern Analysis of ROD1 Gene in Arabidopsis. Data used to create thedigital Northern were obtained from AtGenExpress at the Genevestigatorsite (genevestigator.ethz.ch/). Signal intensities were averaged for allthe stages that are included in this figure; and

FIGS. 9A-9H show, according to particular exemplary aspects, that ROD1possesses the activity of a phosphatidylcholine diacylglycerolcholinephosphotransferase (PDCT). (A) TLC image of CPT assays. (B) TLCimage of PDCT assays. Microsomes of DBY746 (WT) and HJ091 S. cerevisiaecells transformed with p424GPD (V) or p424ROD1 (R) were used, and theTLC solvent system in all experiments is:chloroform/methanol/water=65/25/4 in vol. The substrates areCDP-[14C]Choline and diolein for CPT, and [14C-glycerol]di18:1-DG and PC(0 or 1 mM) for PDCT, respectively. b=boiled microsomal proteins. (C)PDCT activities of ROD1-transformed yeast microsomes in reactions of[14C-glycerol]di18:1-DG with PC (0 or 1 mM), CDP-choline, phosphocholineand lyso-PC, respectively. (D) Microsomes of HJ091 cells transformedwith vector p424GPD (V) or p424ROD1 (R) were incubated with di14:0-PC[14C-Choline] and di18:1-DG for the PDCT assays. (E) The effect of pH onPDCT activity. (F-H) The linearity of the PDCT activity as a function ofincubation time, added microsomal protein and PC, respectively. Datarepresent means and standard deviations of three independent reactions.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Particular aspects provide novel compositions and methods fordifferential regulation of fatty acid unsaturation in seed oil andmembrane lipids, without deleterious alterations in the unsaturation ofmembrane lipid components.

Additional aspects provide compositions and methods for differentialregulation of fatty acid unsaturation in seed oil and membrane lipids ofplants, based on modulation of a previously unknown biosynthetic pathwayinvolving a novel phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) that regulates phosphatidylcholinebiosynthesis in developing oilseed plants (e.g., of Arabidopsis, soybean(Glycine max), canola (Brassica napus or B. rapa), sunflower (Helianthusannuus), etc.).

Further aspects provide inventive PDCT sequences and polypeptidesincluding, for example, mutants (e.g., SEQ ID NOS:4 and 5), variants,deletions, muteins, fusion proteins, and orthologs thereof (collectivelyPDCT proteins).

Yet additional aspects provide plants comprising such PDCT proteins ordevoid or depleted of such PDCT sequences or proteins and/or activities,and methods for generating plants having altered or no PDCT expressionand/or activity, including but not limited to art-recognized methodscomprising mutagenesis, gene-silencing, antisense, siRNA, recombinantDNA, transgenics, etc.). Information about mutagens and mutagenizingseeds or pollen, for example, are presented in the IAEA's Manual onMutation Breeding (IAEA, 1977). In certain embodiments, mutagensiscomprises chemical mutagenesis (e.g., comprising treatment of seeds withethyl methane sulfonate (EMS). Various plant breeding methods are alsouseful in providing inventive plants are discussed in detail hereinbelow.

As described herein below, specific exemplary aspects of the presentinvention provide a genetic and biochemical characterization of anArabidopsis mutant plant with reduced desaturation in seed fatty acids(see Table 1 of EXAMPLE 2 below). The mutant plant, originallyidentified and named as DH4 (7), was indistinguishable from its parentalwild type Col-0 plants grown under standard conditions. Applicantsherein disclose a gene ROD1 (Reduced Oleate Desaturation 1) encoding thePDCT, which mutation in the DH4 Arabidopsis mutant causes reduced oleatedesaturation levels in seed oils. The rod1 allele in DH4 is a singlerecessive Mendelian mutation as determined by genetic analysis. As shownherein (working EXAMPLE 2), the defective PDCT activity in the rod1mutant resulted in impaired transfer of 18:1 fatty acid intophosphatidylcholine (PC) during triacylglycerol synthesis in developingseeds. The results indicate that PDCT is a major factor that regulateslipid flux into phosphatidylcholine, where most fatty acid modificationstake place in oilseeds.

Significantly, compared to the fad2 mutant (5, 7), the fatty acidcomposition change in DH4 is restricted to seed oil.

As described under working EXAMPLE 3 herein below, specific exemplaryaspects of the present invention show that the Arabidopsis mutant rod1locus of DH4 was shown to mediate reduced oleate desaturation in seedoil due to a reduced transfer of 18:1 into PC via de novo synthesis fromdiacylglycerol (DAG).

According to additional aspects, as described in EXAMPLE 4 herein below,fine mapping of the Arabidopsis mutant rod1 of DH4 was performed andAt3g15820 (SEQ ID NO:2) was herein identified as the locus of the rod1mutant (SEQ ID NO:4), and for the first time was shown not only to be aphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), butalso a PDCT that is highly expressed in developing seeds with thehighest level at stage 6 of seed development, which coincides the peakstage of storage deposition.

Significantly, At3g15820 (SEQ ID NO:2) has previously been annotated asa putative type 2 phosphatidic acid phosphatase (PAP2)-like protein.Surprisingly however, upon analysis by Applicants, it did not showstrong homology to known characterized PAP genes in Arabidopsis (AtLPP1,AtLPP2 and AtLPP3) (13, 14), and Applicants have determined herein thatROD1 contains essentially no sequence homology to these true PAP2orthologues, and concluded that ROD1 encodes a different function.

Applicants tested ROD1 for PDCT activity, by expressing the cDNA ofAt3g15820 under control of the inducible GAL1 promoter in adouble-mutant yeast strain HJ091 (17) lacking allCDP-choline:diacylglycerol cholinephosphotransferase activities. Asdetailed herein (EXAMPLE 4), these results indicate that ROD1 does notpossess PA phosphatase activity, and substantially confirms that ROD1rather confers a PDCT activity, which is consistent with the fact thatthe rod1 mutant is defective in PC synthesis in developing seeds.

According to additional aspects, as described in EXAMPLE 5 herein below,ROD1 (At3g15820) orthologs were identified that have significantsequence homology/identity. Tables 2 and 3 of EXAMPLE 5 show nucleotidesimilarity (% identity) and protein sequence similarity (% identity),respectively, for exemplary ROD1 orthologs from Brassica (SEQ ID NO:6;SEQ ID NO:7), Moss (SEQ ID NO:16; SEQ ID NO:17), Spruce (SEQ ID NO:14;SEQ ID NO:15), Grape (SEQ ID NO:12; SEQ ID NO:13), Rice (SEQ ID NO:10;SEQ ID NO:11) and Castor (SEQ ID NO:8; SEQ ID NO:9), showing a range ofnucleic acid identity from about 46 to 80%, and range of proteinsequence identity from about 42 to 85%.

According to further aspects, as described in EXAMPLE 6 herein below,the Brassica napus unigene Bna.6194 is identified as the trueArabidopsis ROD1 (At3g15820) homologue. Applicants named Bna.6194 asBnROD1. Quantitative RT-PCR showed that BnROD1 is highly expressed incanola developing seeds. Brassica napus is an amphidipoid includingBrassica rapa and Brassica oleracea two subgenomes. The sequencealignment also suggested that BnROD1 might be the true homologue ofBrassica rapa unigene Bra. 2038 and Brassica oleracea ES948687.

According to further aspects, as described in EXAMPLE 7 herein below,biological materials (e.g., plant seed oils), as provided for herein,that contain relatively high concentrations of long chain fats withmodest unsaturation provide improved feedstocks for the production ofbiodiesel and related products, as well as food oils.

Specific Preferred Exemplary Embodiments

Particular aspects provide a method for regulation of fatty acidunsaturation in seed oil, comprising: obtaining an oilseed-bearingplant; and modulating the expression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inone or more seeds or developing seeds of the plant, wherein the level,amount, or distribution of fatty acid unsaturation in the seed oil ismodified relative to the seed oil of plants with normal seed expressionof the PDCT. In certain embodiments, modulating the expression oractivity of the at least one PDCT comprises down-regulating theexpression or activity, wherein the level, amount, or distribution offatty acid unsaturation in the seed oil is modified or reduced.Preferably, the method comprises differential regulation of fatty acidunsaturation in seed oil relative to fatty acid unsaturation in one ormore membrane lipids. Preferably, the fatty acid unsaturation in seedoil relative to fatty acid unsaturation in one or more membrane lipidsis differentially reduced in seed oil. In particular embodiments, the atleast one PDCT comprises at least one sequence selected from the groupconsisting of SEQ ID NO:3, a sequence having at least 46, at least 48%,at least 58%, at least 64%, at least 71% or at least 85% amino acidsequence identity therewith, and PDCT-active portions thereof. Incertain embodiments, the at least one PDCT comprises at least onesequence selected from the group consisting of SEQ ID NOS:7, 9, 11, 13,15, 17, and PDCT-active portions thereof. In certain implementation,modulating the expression or activity of the at least one PDCT comprisesthe use of at least one of mutagenesis and recombinant DNA methods,including, but not limited to the use of at least one of gene-silencing,anti-sense methods, siRNA methods, transgenic methods.

Additional aspects provide a method of producing an oil seed-bearingplant or a part thereof, comprising imparting into the germplasm of anoil seed-bearing plant variety a mutation or genetic alteration thatmodifies the expression or activity of at least one PDCT in one or moreseeds or developing seeds of the plant, wherein the level, amount, ordistribution of fatty acid unsaturation in the seed oil is modifiedrelative to the seed oil of plants with normal seed expression of thePDCT. Particular embodiments of the method comprise: providing germplasmof an oil seed-bearing plant variety; treating the germplasm with amutagen to produce a mutagenized germplasm; selecting from themutagenized germplasm an oil seed-bearing plant seed comprising agenotype, caused by the mutagen, that modifies the expression oractivity of at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) in one or more seeds or developingseeds of the plant, wherein the level, amount, or distribution of fattyacid unsaturation in the seed oil is modified relative to the seed oilof plants with normal seed expression of the PDCT; and growing an oilseed-bearing plant from the seed. In particular implementation of themethod, producing a matagenized germplasm comprises producing a mutationof at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) sequence that modifies the expressionor activity thereof in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT. Further embodimentscomprise an oil seed-bearing plant or a part thereof, comprising amutation or genetic alteration that modifies the expression or activityof at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) in one or more seeds or developingseeds of the plant, wherein the level, amount, or distribution of fattyacid unsaturation in the seed oil is modified relative to the seed oilof plants with normal seed expression of the PDCT. While the mutation orgenetic alteration may be one that modulates PDCT expression and/oractivity directly or indirectly, in particular aspects the mutationcomprises a mutation of at least one PDCT sequence that modifies theexpression or activity thereof in one or more seeds or developing seedsof the plant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT. In certain aspects, theoil seed-bearing plant or a part thereof is other than Arabidopsis.Preferably in such plants, modulating the expression or activity of theat least one PDCT comprises down-regulating the expression or activity,wherein the level, amount, or distribution of fatty acid unsaturation inthe seed oil is modified is reduced. Preferably, modulating theexpression or activity comprises differential regulation of fatty acidunsaturation in seed oil relative to fatty acid unsaturation in one ormore membrane lipids. Particular plant embodiments comprise two or moredifferent mutations or genetic alterations that modify the level,amount, or distribution of fatty acid unsaturation in the seed oil,wherein at least one of the two or more different mutations or geneticalterations is a mutation or genetic alteration that modifies theexpression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inone or more seeds or developing seeds of the plant. In particularembodiments of such plants, at least one of the two or more differentmutations is a FAD2 desaturase mutation that reduces or eliminates FAD2activity or amount in the seed or developing seed. In certain aspects,the at least one PDCT comprises at least one sequence selected from thegroup consisting of SEQ ID NO:3, a sequence having at least 46, at least48%, at least 58%, at least 64%, at least 71% or at least 85% amino acidsequence identity therewith, and PDCT-active portions thereof. Inparticular embodiments, the at least one PDCT comprises at least onesequence selected from the group consisting of SEQ ID NOS:7, 9, 11, 13,15, 17, and PDCT-active portions thereof.

Additional aspects provide a seed or true-breeding seed derived from theoil seed-bearing plants or parts thereof as provided for herein.

Further aspects, provide an oil derived from the oil seed-bearing plantsor parts thereof as provided for herein.

Yet additional embodiments provide a fuel, based at least in part on anoil derived from the oil seed-bearing plants or parts thereof asprovided for herein.

Plants and Plant Breeding

Particular aspects provide an oil seed-bearing plant or a part thereof,comprising a mutation or genetic modification that modifies theexpression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inone or more seeds or developing seeds of the plant, wherein the level,amount, or distribution of fatty acid unsaturation in the seed oil ismodified relative to the seed oil of plants with normal seed expressionof the PDCT. While the mutation or genetic modification may be any thatmodifies the PDCT expression and/or activity, in preferred aspect, themutation comprises a mutation of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)sequence that modifies the expression or activity thereof in one or moreseeds or developing seeds of the plant, wherein the level, amount, ordistribution of fatty acid unsaturation in the seed oil is modifiedrelative to the seed oil of plants with normal seed expression of thePDCT.

Various plant breeding methods are also useful in establishing usefulplant varieties based on such mutations or genetic modifications.

Plant Breeding

Additional aspects comprise methods for using, in plant breeding, an oilseed-bearing plant, comprising a mutation that modifies the expressionor activity of at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) (as provided for herein) in one or moreseeds or developing seeds of the plant, wherein the level, amount, ordistribution of fatty acid unsaturation in the seed oil is modifiedrelative to the seed oil of plants with normal seed expression of thePDCT. One such embodiment is the method of crossing a particular PDCTmutant variety with another variety of the plant to form a firstgeneration population of F1 plants. The population of first generationF1 plants produced by this method is also an embodiment of theinvention. This first generation population of F1 plants will comprisean essentially complete set of the alleles of the particular PDCT mutantvariety. One of ordinary skill in the art can utilize either breederbooks or molecular methods to identify a particular F1 plant producedusing the particular PDCT mutant variety, and any such individual plantis also encompassed by this invention. These embodiments also cover useof transgenic or backcross conversions of particular PDCT mutantvarieties to produce first generation F1 plants.

Yet additional aspects comprise a method of developing a particular PDCTmutant-progeny plant comprising crossing a particular PDCT mutantvariety with a second plant and performing a breeding method is also anembodiment of the invention.

General Breeding and Selection Methods

Overview.

Plant breeding is the genetic manipulation of plants. The goal of plantbreeding is to develop new, unique and superior plant varieties. Inpractical application of a plant breeding program, and as discussed inmore detail herein below, the breeder initially selects and crosses twoor more parental lines, followed by repeated ‘selfing’ and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,‘selfing’ and naturally induced mutations. The breeder has no directcontrol at the cellular level, and two breeders will never, therefore,develop exactly the same line. Each year, the plant breeder selects thegermplasm to advance to the next generation. This germplasm may be grownunder unique and different geographical, climatic and soil conditions,and further selections may be made during and at the end of the growingseason.

Proper testing can detect major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, it is desirable that this a demand for anew variety. The new variety should optimally be compatible withindustry standards, or create a new market. The introduction of a newvariety may incur additional costs to the seed producer, the grower,processor and consumer, for special advertising and marketing, alteredseed and commercial production practices, and new product utilization.The testing preceding release of a new variety should take intoconsideration research and development costs as well as technicalsuperiority of the final variety. Ideally, it should also be feasible toproduce seed easily and economically.

The term ‘homozygous plant’ is hereby defined as a plant with homozygousgenes at 95% or more of its loci.

The term “inbred” as used herein refers to a homozygous plant or acollection of homozygous plants.

Choice of Breeding or Selection Methods.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F1 hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. The complexity of inheritance also influences choice of thebreeding method. Breeding generally starts with cross-hybridizing twogenotypes (a “breeding cross”), each of which may have one or moredesirable characteristics that is lacking in the other or whichcomplements the other. If the two original parents do not provide allthe desired characteristics, other sources can be included by makingmore crosses. In each successive filial generation (e.g., F1→F2; F2→F3;F3→F4; F4→F5, etc.), plants are ‘selfed’ to increase the homozygosity ofthe line. Typically in a breeding program five or more generations ofselection and ‘selfing’ are practiced to obtain a homozygous plant. Eachplant breeding program should include a periodic, objective evaluationof the efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulvarieties produced per unit of input (e.g., per year, per dollarexpended, etc.).

Backcross Conversion

An additional embodiment comprises or is a backcross conversion of anoil seed-bearing plant, comprising a mutation that modifies theexpression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (asprovided for herein) in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT. A backcross conversionoccurs when DNA sequences are introduced through traditional(non-transformation) breeding techniques, such as backcrossing. DNAsequences, whether naturally occurring or transgenes, may be introducedusing these traditional breeding techniques. Desired traits transferredthrough this process include, but are not limited to nutritionalenhancements, industrial enhancements, disease resistance, insectresistance, herbicide resistance, agronomic enhancements, grain qualityenhancement, waxy starch, breeding enhancements, seed productionenhancements, and male steriltiy. A further embodiment comprises or is amethod of developing a backcross conversion plant that involves therepeated backcrossing to such PDCT mutations. The number of backcrossesmade may be 2, 3, 4, 5, 6 or greater, and the specific number ofbackcrosses used will depend upon the genetics of the donor parent andwhether molecular markers are utilized in the backcrossing program.

Essentially Derived Varieties

Another embodiment of the invention is an essentially derived variety ofan oil seed-bearing plant, comprising a mutation that modifies theexpression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (asprovided for herein) in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT. As determined by theUPOV Convention, essentially derived varieties may be obtained forexample by the selection of a natural or induced mutant, or of asomaclonal variant, the selection of a variant individual from plants ofthe initial variety, backcrossing, or transformation by geneticengineering. An essentially derived variety of such PDCT mutants isfurther defined as one whose production requires the repeated usethereof, or is predominately derived from genotype of a particular PDCTmutant. International Convention for the Protection of New Varieties ofPlants, as amended on Mar. 19, 1991, Chapter V, Article 14, Section5(c).

DNA Constructs

The present invention also contemplates the fabrication of DNAconstructs (e.g., expression vectors, recombination vectors, anti-senseconstructs, siRNA constructs, etc.) comprising the isolated nucleic acidsequence containing the genetic element and/or coding sequence from thedisclosed PDCT mutant varieties operatively linked to plant geneexpression control sequences. “DNA constructs” are defined herein to beconstructed (not naturally-occurring) DNA molecules useful forintroducing DNA into host cells, and the term includes chimeric genes,expression cassettes, and vectors.

As used herein “operatively linked” refers to the linking of DNAsequences (including the order of the sequences, the orientation of thesequences, and the relative spacing of the various sequences) in such amanner that the encoded protein is expressed. Methods of operativelylinking expression control sequences to coding sequences are well knownin the art. See, e.g., Maniatis, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., 1982; and Sambrook, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989.

“Expression control sequences” are DNA sequences involved in any way inthe control of transcription or translation. Suitable expression controlsequences and methods of making and using them are well known in theart.

The expression control sequences preferably include a promoter. Thepromoter may be inducible or constitutive. It may benaturally-occurring, may be composed of portions of variousnaturally-occurring promoters, or may be partially or totally synthetic.Guidance for the design of promoters is provided by studies of promoterstructure, such as that of Harley and Reynolds, Nucleic Acids Res., 15,2343-2361, 1987. Also, the location of the promoter relative to thetranscription start may be optimized. See, e.g., Roberts, et al., Proc.Natl. Acad. Sci. USA, 76:760-764, 1979.

Many suitable promoters for use in plants are well known in the art. Forinstance, suitable constitutive promoters for use in plants include thepromoters of plant viruses, such as the peanut chlorotic streakcaulimovirus (PC1SV) promoter (U.S. Pat. No. 5,850,019); the 35S and 19Spromoter from cauliflower mosaic virus (CaMV) (Odell, et al., I313:3810-812, 1985); promoters of the Chlorella virus methyltransferasegenes (U.S. Pat. No. 5,563,328); the full-length transcript promoterfrom figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promotersfrom such genes as rice actin (McElroy, et al., Plant Cell 2:163-171(1990)), ubiquitin (Christiansen, et al., Plant Mol. Biol. 12:619-632,1989), and (Christiansen, et al., Plant Mol. Biol. 18: 675-689, 1992),pEMU (Last, et al., Theor. Appl. Genet. 81:581-588, 1991), MAS (Velten,et al., Embo J. 3:2723-2730, 1984), wheat histone (Lepetit, et al., Mol.Gen. Genet. 231:276-285, 1992), and Atanassova, et al., Plant Journal2:291-300, 1992), Brassica napus ALS3 (International Publication No. WO1997/41228); and promoters of various Agrobacterium genes (see U.S. Pat.Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

Suitable inducible promoters for use in plants include: the promoterfrom the ACE1 system which responds to copper (Mett, et al., Proc. Natl.Acad. Sci. 90:4567-4571, 1993): the promoter of the wheat In 2 genewhich responds to benzenesulfonomide herbicide safeners (U.S. Pat. No.5,364,780 and Gatz, et al., Mol. Gen. Genet. 243:32-38, 1994), and thepromoter of the Tet repressor from Tn10 (Gatz, et al., Mol. Gen. Genet.227:229-237, 1991). According to one embodiment, the promoter for use inplants is one that responds to an inducing agent to which plantsnormally do not respond. An exemplary inducible promoter of this type isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucosteroid hormone (Schena, et al.,Proc. Natl. Acad. Sci. 88:10421, 1991) or the application of a chimerictranscription activator, XVE, for use in an estrogen receptor-basedinducible plant expression system activated by estradiol (Zou, et al.,Plant J. 24 265-273, 2000). Other inducible promoters for use in plantsare described in European Patent No. 332104, International PublicationNo. WO 1993/21334 and International Publication No. WO 1997/06269, anddiscussed in Gatz and Lenk Trends Plant Sci., 3:352-358, 1998, and Zouand Chua, Curr. Opin. Biotechnol., 11:146-151, 2000. Finally, promoterscomposed of portions of other promoters and partially or totallysynthetic promoters can be used. See, e.g., Ni, et al., Plant J.7:661-676, 1995, and International Publication No. WO 1995/14098, whichdescribes such promoters for use in plants.

The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters that do notinclude them. Suitable enhancer elements for use in plants include thePC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV 35S enhancerelement (U.S. Pat. Nos. 5,106,739 and 5,164,316), and the FMV enhancerelement (Maiti, et al., Transgenic Res., 6:143-156, 1997). See also,International Publication No. WO 1996/23898 and Enhancers and EukaryoticExpression (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1983).

For efficient expression, the coding sequences are preferably alsooperatively linked to a 3′ untranslated sequence. The 3′ untranslatedsequence will preferably include a transcription termination sequenceand a polyadenylation sequence. The 3′ untranslated region can beobtained from the flanking regions of genes from Agrobacterium, plantviruses, plants and other eukaryotes. Suitable 3′ untranslated sequencesfor use in plants include those of the cauliflower mosaic virus 35Sgene, the phaseolin seed storage protein gene, the pearibulose-1,5-bisphosphate carboxylase small subunit E9 gene, the wheat7S storage protein gene, the octopine synthase gene, and the nopalinesynthase gene.

A 5′ untranslated leader sequence can also be optionally employed. The5′ untranslated leader sequence is the portion of an mRNA that extendsfrom the 5′ CAP site to the translation initiation codon. This region ofthe mRNA is necessary for translation initiation in plants and plays arole in the regulation of gene expression. Suitable 5′ untranslatedleader sequence for use in plants includes those of alfalfa mosaicvirus, cucumber mosaic virus coat protein gene, and tobacco mosaicvirus.

The DNA construct may be a ‘vector.’ The vector may contain one or morereplication systems which allow it to replicate in host cells.Self-replicating vectors include plasmids, cosmids and virus vectors.Alternatively, the vector may be an integrating vector which allows theintegration into the host cell's chromosome of the DNA sequence encodingthe root-rot resistance gene product. The vector desirably also hasunique restriction sites for the insertion of DNA sequences. If a vectordoes not have unique restriction sites it may be modified to introduceor eliminate restriction sites to make it more suitable for furthermanipulation.

Vectors suitable for use in expressing the nucleic acids, which whenexpressed in a plant modulate the expression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (asprovided for herein) in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT, include but are notlimited to pMON979, pMON977, pMON886, pCaMVCN, and vectors derived fromthe tumor inducing (Ti) plasmid of Agrobacterium tumefaciens describedby Rogers, et al., Meth. Enzymol., 153:253-277, 1987. The nucleic acidis inserted into the vector such that it is operably linked to asuitable plant active promoter. Suitable plant active promoters for usewith the nucleic acids include, but are not limited to CaMV35S, ACTIN,FMV35S, NOS and PCSLV promoters. The vectors comprising the nucleic acidcan be inserted into a plant cell using a variety of known methods. Forexample, DNA transformation of plant cells include but are not limitedto Agrobacterium-mediated plant transformation, protoplasttransformation, electroporation, gene transfer into pollen, injectioninto reproductive organs, injection into immature embryos and particlebombardment. These methods are described more fully in U.S. Pat. No.5,756,290, and in a particularly efficient protocol for wheat describedin U.S. Pat. No. 6,153,812, and the references cited therein.Site-specific recombination systems can also be employed to reduce thecopy number and random integration of the nucleic acid into the plantgenome. For example, the Cre/lox system can be used to immediate loxsite-specific recombination in plant cells. This method can be found atleast in Choi, et al., Nuc. Acids Res. 28:B19, 2000).

Transgenes:

Molecular biological techniques allow the isolation and characterizationof genetic elements with specific functions, such as encoding specificprotein products. Scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genetic elements, or additional, or modified versions ofnative or endogenous genetic elements in order to alter the traits of aplant in a specific manner. Any DNA sequences, whether from a differentspecies or from the same species, that are inserted into the genomeusing transformation are referred to herein collectively as“transgenes.” Several methods for producing transgenic plants have beendeveloped, and the present invention, in particular embodiments, alsorelates to transformed versions of the genotypes of the invention and/ortransformed versions comprising one or more transgenes modify directlyor indirectly the expression or activity of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (asprovided for herein) in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber, et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993), pages 89-119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements. Various geneticelements can be introduced into the plant genome using transformation.These elements include but are not limited to genes; coding sequences(in sense or anti-sense orientation); inducible, constitutive, andtissue specific promoters; enhancing sequences; and signal and targetingsequences.

A genetic trait which has been engineered into a particular plant usingtransformation techniques could be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove a transgene from a transformed oil seed-bearing plant to an eliteplant variety and the resulting progeny would comprise a transgene. Asused herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context. The term “breedingcross” excludes the processes of selfing or sibbing.

With transgenic plants according to the present invention, a foreignprotein and/or and modified expression of an endogenous protein orproduct (e.g., oil) can be produced in commercial quantities. Thus,techniques for the selection and propagation of transformed plants,which are well understood in the art, yield a plurality of transgenicplants which are harvested in a conventional manner, and a plant productcan then can be extracted from a tissue of interest or from totalbiomass. Protein and oil extraction from plant biomass can beaccomplished by known methods which are discussed, for example, by Heneyand Orr, Anal. Biochem. 114:92-96, 1981.

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is canola (e.g., Brassica napusor B. rapa), soybean (e.g., Glycine max), or sunflower (e.g., Helianthusannuus). In another preferred embodiment, the biomass of interest isseed. A genetic map can be generated, primarily via conventional RFLP,PCR, and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology 269-284 (CRC Press, Boca Raton, 1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Introduction of Transgenes of Agronomic Interest by Transformation

Agronomic genes can be expressed in transformed plants. For example,plants can be genetically engineered to express various phenotypes ofagronomic interest, or, alternatively, transgenes can be introduced intoa plant by breeding with a plant that has the transgene. Through thetransformation of plant, the expression of genes can be modulated toenhance disease resistance, insect resistance, herbicide resistance,water stress tolerance and agronomic traits as well as seed qualitytraits. Transformation can also be used to insert DNA sequences whichcontrol or help control male-sterility. DNA sequences native toparticular plants as well as non-native DNA sequences can be transformedand used to modulate levels of native or non-native proteins. Anti-sensetechnology, siRNA technology, various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into theparticular genome for the purpose of modulating the expression ofproteins. Many exemplary genes implicated in this regard are known inthe art.

Variants of Phosphatidylcholine:Diacylglycerol Cholinephosphotransferase(PDCT) Nucleic Acids and Proteins

As used herein, a “biological activity” refers to a function of apolypeptide including but not limited to complexation, dimerization,multimerization, receptor-associated ligand binding and/or endocytosis,receptor-associated protease activity, phosphorylation,dephosphorylation, autophosphorylation, ability to form complexes withother molecules, ligand binding, catalytic or enzymatic activity,activation including auto-activation and activation of otherpolypeptides, inhibition or modulation of another molecule's function,stimulation or inhibition of signal transduction and/or cellularresponses such as cell proliferation, migration, differentiation, andgrowth, degradation, membrane localization, and membrane binding. Abiological activity can be assessed by assays described herein and byany suitable assays known to those of skill in the art, including, butnot limited to in vitro assays, including cell-based assays, in vivoassays, including assays in animal models for particular diseases.

Preferably, the phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT), or variants thereof) comprises anamino acid sequence of SEQ ID NO:3 (or of SEQ ID NO:5 having from 1, toabout 3, to about 5, to about 10, or to about 20 conservative amino acidsubstitutions), or a fragment of a sequence of SEQ ID NO:3 (or of SEQ IDNO:5 having from 1, to about 3, to about 5, to about 10, or to about 20conservative amino acid substitutions). Preferably,phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), orvariant thereof, comprises a sequence of SEQ ID NO:2, or SEQ ID NO:5, ora conservative amino acid substitution variant thereof.

Functional phosphatidylcholine:diacylglycerol cholinephosphotransferase(PDCT), variants are those proteins that display (or lack) one or moreof the biological activities of phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT).

As used herein, the term “wild type ROD1 or PDCT”, means a naturallyoccurring ROD1 or PDCT allele found within plants which encodes afunctional ROD1 or PDCT protein. In contrast, the term “mutant ROD1 orPDCT”, as used herein, refers to an ROD1 or PDCT allele, which does notencode a functional ROD1 or PDCT protein, i.e., an ROD1 or PDCT alleleencoding a non-functional ROD1 or PDCT protein, which, as used herein,refers to an ROD1 or PDCT protein having no biological activity or asignificantly reduced biological activity as compared to thecorresponding wild-type functional ROD1 or PDCT protein, or encoding noROD1 or PDCT protein at all. Such a “mutant ROD1 or PDCT allele” (alsocalled “full knock-out” or “null” allele) is a wild-type ROD1 or PDCTallele, which comprises one or more mutations in its nucleic acidsequence, whereby the mutation(s) preferably result in a significantlyreduced (absolute or relative) amount of functional ROD1 or PDCT proteinin the cell in vivo. Mutant alleles of the ROD1 or PDCT protein-encodingnucleic acid sequences are designated as “rod1 or pdct” herein. Mutantalleles can be either “natural mutant” alleles, which are mutant allelesfound in nature (e.g., produced spontaneously without human applicationof mutagens) or induced mutant” alleles, which are induced by humanintervention, e.g., by mutagenesis.

As used herein, the term “wild type ROD1 or PDCT”, means a naturallyoccurring ROD1 or PDCT allele found within plants which encodes afunctional ROD1 or PDCT protein. In contrast, in particular aspects, theterm “mutant ROD1 or PDCT”, as used herein, refers to an ROD1 or PDCTallele, which does not encode a functional ROD1 or PDCT protein, i.e.,an ROD1 or PDCT allele encoding a non-functional ROD1 or PDCT protein,which, as used herein, refers to an ROD1 or PDCT protein having nobiological activity or a significantly reduced biological activity ascompared to the corresponding wild-type functional ROD1 or PDCT protein,or encoding no ROD1 or PDCT protein at all. Such a “mutant ROD1 or PDCTallele” (also called “full knock-out” or “null” allele) is a wild-typeROD1 or PDCT allele, which comprises one or more mutations in itsnucleic acid sequence, whereby the mutation(s) preferably result in asignificantly reduced (absolute or relative) amount of functional ROD1or PDCT protein in the cell in vivo. Mutant alleles of the ROD1 or PDCTprotein-encoding nucleic acid sequences are designated as “rod1 or pdct”herein. Mutant alleles can be either “natural mutant” alleles, which aremutant alleles found in nature (e.g., produced spontaneously withouthuman application of mutagens) or induced mutant” alleles, which areinduced by human intervention, e.g., by mutagenesis.

Variants of phosphatidylcholine:diacylglycerol cholinephosphotransferase(PDCT) have utility for aspects of the present invention. Variants canbe naturally or non-naturally occurring. Naturally occurring variants(e.g., polymorphisms) are found in various species and comprise aminoacid sequences which are substantially identical to the amino acidsequence shown in SEQ ID NO:3 or SEQ ID NO:5. Species homologs of theprotein can be obtained using subgenomic polynucleotides of theinvention, as described below, to make suitable probes or primers forscreening cDNA expression libraries from other species, such as mice,monkeys, yeast, or bacteria, identifying cDNAs which encode homologs ofthe protein, and expressing the cDNAs as is known in the art. Orthologsare provided for herein.

Non-naturally occurring variants which retain (or lack) substantiallythe same biological activities as naturally occurring protein variantsare also included here. Preferably, naturally or non-naturally occurringvariants have amino acid sequences which are at least 85%, 90%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to the amino acidsequence shown in SEQ ID NOS:3 or 5. More preferably, the molecules areat least 98%, 99% or greater than 99% identical. Percent identity isdetermined using any method known in the art. A non-limiting example isthe Smith-Waterman homology search algorithm using an affine gap searchwith a gap open penalty of 12 and a gap extension penalty of 1. TheSmith-Waterman homology search algorithm is taught in Smith andWaterman, Adv. Appl. Math. 2:482-489, 1981.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are generally in the“L” isomeric form. Residues in the “D” isomeric form can be substitutedfor any L-amino acid residue, as long as the desired functional propertyis retained by the polypeptide. NH2 refers to the free amino grouppresent at the amino terminus of a polypeptide. COOH refers to the freecarboxy group present at the carboxyl terminus of a polypeptide. Inkeeping with standard polypeptide nomenclature described in J. Biol.Chem., 243:3552-59 (1969) and adopted at 37 C.F.R. §§ 1.821-1.822,abbreviations for amino acid residues are shown in Table 2:

TABLE 2 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp aspartic acid N Asn Asparagines B Asx Asn and/or Asp C Cys CysteineX Xaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by a formula have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is defined to include the amino acids listedin the Table of Correspondence and modified and unusual amino acids,such as those referred to in 37 C.F.R. §§ 1.821-1.822, and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or to anamino-terminal group such as NH₂ or to a carboxyl-terminal group such asCOOH.

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological or immunologicalactivity can be found using computer programs well known in the art,such as DNASTAR software. Preferably, amino acid changes in the proteinvariants disclosed herein are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. Preferably, amino acid changes in thephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptide variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting variant. Properties and functionsof phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptide protein or polypeptide variants are of the same type as aprotein comprising the amino acid sequence encoded by the nucleotidesequence shown in SEQ ID NOS:3 and 5, although the properties andfunctions of variants can differ in degree.

Variants of the phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) polypeptide disclosed herein includeglycosylated forms, aggregative conjugates with other molecules, andcovalent conjugates with unrelated chemical moieties (e.g., pegylatedmolecules). Covalent variants can be prepared by linking functionalitiesto groups which are found in the amino acid chain or at the N- orC-terminal residue, as is known in the art. Variants also includeallelic variants, species variants, and muteins. Truncations ordeletions of regions which do or do not affect functional activity ofthe proteins are also variants. Covalent variants can be prepared bylinking functionalities to groups which are found in the amino acidchain or at the N- or C-terminal residue, as is known in the art.

A subset of mutants, called muteins, is a group of polypeptides in whichneutral amino acids, such as serines, are substituted for cysteineresidues which do not participate in disulfide bonds. These mutants maybe stable over a broader temperature range than native secreted proteins(see, e.g., Mark, et al., U.S. Pat. No. 4,959,314).

It will be recognized in the art that some amino acid sequences of thephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptides of the invention can be varied without significant effecton the structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there arecritical areas on the protein which determine activity. In general, itis possible to replace residues that form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein. Thereplacement of amino acids can also change the selectivity of ligandbinding to cell surface receptors (Ostade, et al., Nature 361:266-268,1993). Thus, the phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) polypeptides of the present inventionmay include one or more amino acid substitutions, deletions oradditions, either from natural mutations or human manipulation.

Amino acids in the phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) polypeptides of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as binding to a natural or synthetic bindingpartner. Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.Biol. 224:899-904 (1992) and de Vos, et al. Science 255:306-312 (1992)).

As indicated, changes in particular aspects are preferably of a minornature, such as conservative amino acid substitutions that do notsignificantly affect the folding or activity of the protein. Of course,the number of amino acid substitutions a skilled artisan would makedepends on many factors, including those described above. Otherembodiments comprise non-conservative substitutions. Generally speaking,the number of substitutions for any givenphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5, or 3.

Fusion Proteins

Fusion proteins comprising proteins or polypeptide fragments ofphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptide can also be constructed. Fusion proteins are useful forgenerating antibodies against amino acid sequences and for use invarious targeting and assay systems. For example, fusion proteins can beused to identify proteins which interact with aphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)polypeptide of the invention or which interfere with its biologicalfunction. Physical methods, such as protein affinity chromatography, orlibrary-based assays for protein-protein interactions, such as the yeasttwo-hybrid or phage display systems, can also be used for this purpose.Such methods are well known in the art and can also be used as drugscreens. Fusion proteins comprising a signal sequence can be used.

A fusion protein comprises two protein segments fused together by meansof a peptide bond. Amino acid sequences for use in fusion proteins ofthe invention can be utilize the amino acid sequence shown in SEQ IDNOS:3 or 5, or can be prepared from biologically active variants of SEQID NOS:3 or 5, such as those described above. The first protein segmentcan include of a full-length phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) polypeptide. Other first proteinsegments can consist of about functional portions of SEQ ID NOS:3 and 5.

The second protein segment can be a full-length protein or a polypeptidefragment. Proteins commonly used in fusion protein construction includeβ-galactosidase, β-glucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horseradish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags can be used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GAL4 DNA binding domain fusions, and virus proteinfusions.

These fusions can be made, for example, by covalently linking twoprotein segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be use to prepare fusion proteins,for example, by making a DNA construct which comprises a coding regionfor the protein sequence of SEQ ID NOS:3 and 5 in proper reading framewith a nucleotide encoding the second protein segment and expressing theDNA construct in a host cell, as is known in the art. Many kits forconstructing fusion proteins are available from companies that supplyresearch labs with tools for experiments, including, for example,Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.),Clontech (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz,Calif.), MBL International Corporation (MIC; Watertown, Mass.), andQuantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Nucleic Acid Sequences Encoding Mutant ROD1 or PDCT Proteins

Nucleic acid sequences comprising one or more nucleotide deletions,insertions or substitutions relative to the wild type nucleic acidsequences are another embodiment of the invention, as are fragments ofsuch mutant nucleic acid molecules. Such mutant nucleic acid sequences(referred to as ROD1 or PDCT sequences) can be generated and 1identified using various known methods, as described further below.Again, such nucleic acid molecules are provided both in endogenous formand in isolated form. In one embodiment, the mutation(s) result in oneor more changes (deletions, insertions and/or substitutions) in theamino acid sequence of the encoded ROD1 or PDCT protein (i.e., it is nota “silent mutation”). In another embodiment, the mutation(s) in thenucleic acid sequence result in a significantly reduced or completelyabolished biological activity of the encoded ROD1 or PDCT proteinrelative to the wild type protein.

The nucleic acid molecules may, thus, comprise one or more mutations,such as:

(a) a “missense mutation”, which is a change in the nucleic acidsequence that results in the substitution of an amino acid for anotheramino acid;

(b) a “nonsense mutation” or “STOP codon mutation”, which is a change inthe nucleic acid sequence that results in the introduction of apremature STOP codon and thus the termination of translation (resultingin a truncated protein); plant genes contain the translation stop codons“TGA” (UGA in RNA), “TAA” (UAA in RNA) and “TAG” (UAG in RNA); thus anynucleotide substitution, insertion, deletion which results in one ofthese codons to be in the mature mRNA being translated (in the readingframe) will terminate translation.

(c) an “insertion mutation” of one or more amino acids, due to one ormore codons having been added in the coding sequence of the nucleicacid;

(d) a “deletion mutation” of one or more amino acids, due to one or morecodons having been deleted in the coding sequence of the nucleic acid;

(e) a “frameshift mutation”, resulting in the nucleic acid sequencebeing translated in a different frame downstream of the mutation. Aframeshift mutation can have various causes, such as the insertion,deletion or duplication of one or more nucleotides.

It is desired that the mutation(s) in the nucleic acid sequencepreferably result in a mutant protein comprising significantly reducedor no biological activity in vivo or in the production of no protein.Basically, any mutation which results in a protein comprising at leastone amino acid insertion, deletion and/or substitution relative to thewild type protein can lead to significantly reduced or no biologicalactivity. It is, however, understood that mutations in certain parts ofthe protein are more likely to result in a reduced function of themutant ROD1 or PDCT protein, such as mutations leading to truncatedproteins, whereby significant portions of the functional domains arelacking.

Thus in one embodiment, nucleic acid sequences comprising one or more ofany of the types of mutations described above are provided. In anotherembodiment, ROD1 or PDCT sequences comprising one or more stop codon(nonsense) mutations, one or more missense mutations and/or one or moreframeshift mutations are provided. Any of the above mutant nucleic acidsequences are provided per se (in isolated form), as are plants andplant parts comprising such sequences endogenously. In the tables hereinbelow the most preferred ROD1 or PDCT alleles are described.

A nonsense mutation in an ROD1 or PDCT allele, as used herein, is amutation in an ROD1 or PDCT allele whereby one or more translation stopcodons are introduced into the coding DNA and the corresponding mRNAsequence of the corresponding wild 1 type ROD1 or PDCT allele.Translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG(UAG). Thus, any mutation (deletion, insertion or substitution) thatleads to the generation of an in-frame stop codon in the coding sequencewill result in termination of translation and truncation of the aminoacid chain. In one embodiment, a mutant ROD1 or PDCT allele comprising anonsense mutation is an ROD1 or PDCT allele wherein an in-frame stopcodon is introduced in the ROD1 or PDCT codon sequence by a singlenucleotide substitution, such as the mutation of CAG to TAG, TGG to TAG,TGG to TGA, or CAA to TAA. In another embodiment, a mutant ROD1 or PDCTallele comprising a nonsense mutation is an ROD1 or PDCT allele whereinan in-frame stop codon is introduced in the ROD1 or PDCT codon sequenceby double nucleotide substitutions, such as the mutation of CAG to TAA,TGG to TAA, or CGG to TAG or TGA. In yet another embodiment, a mutantROD1 or PDCT allele comprising a nonsense mutation is an ROD1 or PDCTallele wherein an in-frame stop codon is introduced in the ROD1 or PDCTcodon sequence by triple nucleotide substitutions, such as the mutationof CGG to TAA. The truncated protein lacks the amino acids encoded bythe coding DNA downstream of the mutation (i.e., the C-terminal part ofthe ROD1 or PDCT protein) and maintains the amino acids encoded by thecoding DNA upstream of the mutation (i.e., the N-terminal part of theROD1 or PDCT protein).

The Tables herein below describe a range of possible nonsense mutationsin the ROD1 or PDCT sequences provided herein:

TABLE 1a Potential STOP codon mutations in At-ROD1 (SEQ ID NO: 1) geneposition position initial codon stop_codon At_rod 1 199 201 TGG TAGAt_rod 1 199 201 TGG TAA At_rod 1 199 201 TGG TGA At_rod 1 226 228 TGGTGA At_rod 1 226 228 TGG TAG At_rod 1 226 228 TGG TAA At_rod 1 265 267TGG TGA At_rod 1 265 267 TGG TAA At_rod 1 265 267 TGG TAG At_rod 1 325327 CAG TAG At_rod 1 325 327 CAG TAA At_rod 1 457 459 CAA TAA At_rod 1475 477 TGG TAA At_rod 1 475 477 TGG TAG At_rod 1 475 477 TGG TGA At_rod1 481 483 TGG TAG At_rod 1 481 483 TGG TGA At_rod 1 481 483 TGG TAAAt_rod 1 496 498 CGA TGA At_rod 1 496 498 CGA TAA At_rod 1 502 504 CGATGA At_rod 1 502 504 CGA TAA At_rod 1 562 564 CAG TAA At_rod 1 562 564CAG TAG At_rod 1 577 579 CAG TAG At_rod 1 577 579 CAG TAA At_rod 1 691693 CAG TAG At_rod 1 691 693 CAG TAA At_rod 1 736 738 CAG TAG At_rod 1736 738 CAG TAA

TABLE 2b Potential STOP codon mutations in ROD1 orthologue from Brassicanapus ( SEQ ID NO: 19) position position initial codon stop_codon 166168 TGG TGA 166 168 TGG TAG 166 168 TGG TAA 205 207 TGG TGA 205 207 TGGTAA 205 207 TGG TAG 265 267 CAG TAG 265 267 CAG TAA 397 399 CAA TAA 415417 TGG TAG 415 417 TGG TGA 415 417 TGG TAA 421 423 TGG TAG 421 423 TGGTGA 421 423 TGG TAA 442 444 CGA TGA 442 444 CGA TAA 502 504 CAG TAA 502504 CAG TAG 517 519 CAG TAG 517 519 CAG TAA 631 633 CAG TAA 631 633 CAGTAG 676 678 CAA TAA

Obviously, mutations are not limited to the ones shown in the abovetables and it is understood that analogous STOP mutations may be presentin ROD1 or PDCT alleles other than those depicted in the sequencelisting and referred to in the tables above.

A missense mutation in an ROD1 or PDCT allele, as used herein, is anymutation (deletion, insertion or substitution) in an ROD1 or PDCT allelewhereby one or more codons are changed in the coding DNA and thecorresponding mRNA sequence of the corresponding wild type ROD1 or PDCTallele, resulting in the substitution of one or more amino acids in thewild type ROD1 or PDCT protein for one or more other amino acids in themutant ROD1 or PDCT protein.

A frameshift mutation in an ROD1 or PDCT allele, as used herein, is amutation (deletion, insertion, duplication, and the like) in an ROD1 orPDCT allele that results in the nucleic acid sequence being translatedin a different frame downstream of the mutation.

Downregulation of ROD1:

Several methods are available in the art to produce a silencing RNAmolecule, i.e., an RNA molecule which when expressed reduces theexpression of a particular gene or group of genes, including theso-called “sense” or “antisense” RNA technologies.

Antisense Technology.

Thus in one embodiment, the inhibitory RNA molecule encoding chimericgene is based on the so-called antisense technology. In other words, thecoding region of the chimeric gene comprises a nucleotide sequence of atleast 19 or 20 consecutive nucleotides of the complement of thenucleotide sequence of the ROD1 or an orthologue thereof. Such achimeric gene may be constructed by operably linking a DNA fragmentcomprising at least 19 or 20 nucleotides from ROD1 encoding gene or anorthologue thereof, isolated or identified as described elsewhere inthis application, in inverse orientation to a plant expressible promoterand 3′ end formation region involved in transcription termination andpolyadenylation.

Co-Suppression Technology.

In another embodiment, the inhibitory RNA molecule encoding chimericgene is based on the so-called co-suppression technology. In otherwords, the coding region of the chimeric gene comprises a nucleotidesequence of at least 19 or 20 consecutive nucleotides of the nucleotidesequence of the ROD1 encoding gene or an orthologue thereof (or in someembodiments, the fiber selective β-1,3 endoglucanase gene). Such achimeric gene may be constructed by operably linking a DNA fragmentcomprising at least 19 or 20 nucleotides from the ROD1 encoding gene oran orthologue thereof, in direct orientation to a plant expressiblepromoter and 3′ end formation region involved in transcriptiontermination and polyadenylation.

The efficiency of the above mentioned chimeric genes in reducing theexpression of the the ROD1 encoding gene or an orthologue thereof may befurther enhanced by the inclusion of DNA element which result in theexpression of aberrant, unpolyadenylated inhibitory RNA molecules orresults in the retention of the inhibitory RNA molecules in the nucleusof the cells. One such DNA element suitable for that purpose is a DNAregion encoding a self-splicing ribozyme, as described inPCT/IB2000/01133, published as WO 2001/012824 (incorporated herein byreference in its entirety and particularly for its teachings onself-splicing ribozymes). Another such DNA element suitable for thatpurpose is a DNA region encoding an RNA nuclear localization orretention signal, as described in PCT/AU2003/00292, published as WO2003/076619 (incorporated by reference).

Double-Stranded RNA (dsRNA) or Interfering RNA (RNAi).

A convenient and very efficient way of downregulating the expression ofa gene of interest uses so-called double-stranded RNA (dsRNA) orinterfering RNA (RNAi), as described e.g., in WO 1999/53050(incorporated herein by reference in its entirety and particularly forits teachings on RNAi)). In this technology, an RNA molecule isintroduced into a plant cell, whereby the RNA molecule is capable offorming a double stranded RNA region over at least about 19 to about 21nucleotides, and whereby one of the strands of this double stranded RNAregion is about identical in nucleotide sequence to the target gene(“sense region”), whereas the other strand is about identical innucleotide sequence to the complement of the target gene or of the senseregion (“antisense region”). It is expected that for silencing of thetarget gene expression, the nucleotide sequence of the 19 consecutivenucleotide sequences may have one mismatch, or the sense and antisenseregion may differ in one nucleotide. To achieve the construction of suchRNA molecules or the encoding chimeric genes, use can be made of thevector as described in WO 2002/059294.

Thus, in one embodiment of the invention, a method for regulating fattyacid unsaturation in seed oil, is provided comprising the step ofintroducing a chimeric gene into a cell of the plant, wherein thechimeric gene comprises the following operably linked DNA elements:

-   -   (a) a plant expressible promoter;    -   (b) a transcribed DNA region, which when transcribed yields a        double-stranded RNA molecule capable of reducing specifically        the expression of ROD1 or an orthologue thereof, and the RNA        molecule comprising a first and second RNA region wherein        -   i) the first RNA region comprises a nucleotide sequence of            at least 19 consecutive nucleotides having at least about            94% sequence identity to the nucleotide sequence of ROD1 or            of an orthologue thereof;        -   ii) the second RNA region comprises a nucleotide sequence            complementary to the at least 19 consecutive nucleotides of            the first RNA region;        -   iii) the first and second RNA region are capable of            base-pairing to form a double stranded RNA molecule between            at least the 19 consecutive nucleotides of the first and            second region; and

(c) a 3′ end region comprising transcription termination andpolyadenylation signals functioning in cells of the plant.

The length of the first or second RNA region (sense or antisense region)may vary from about 19 nucleotides (nt) up to a length equaling thelength (in nucleotides) of the endogenous gene involved in calloseremoval. The total length of the sense or antisense nucleotide sequencemay thus be at least at least 25 nt, or at least about 50 nt, or atleast about 100 nt, or at least about 150 nt, or at least about 200 nt,or at least about 500 nt. It is expected that there is no upper limit tothe total length of the sense or the antisense nucleotide sequence.However for practical reasons (such as e.g., stability of the chimericgenes) it is expected that the length of the sense or antisensenucleotide sequence should not exceed 5000 nt, particularly should notexceed 2500 nt and could be limited to about 1000 nt.

It will be appreciated that the longer the total length of the sense orantisense region, the less stringent the requirements for sequenceidentity between these regions and the corresponding sequence in ROD1gene and orthologues or their complements. Preferably, the nucleic acidof interest should have a sequence identity of at least about 75% withthe corresponding target sequence, particularly at least about 80%, moreparticularly at least about 85%, quite particularly about 90%,especially about 95%, more especially about 100%, quite especially beidentical to the corresponding part of the target sequence or itscomplement. However, it is preferred that the nucleic acid of interestalways includes a sequence of about 19 consecutive nucleotides,particularly about 25 nt, more particularly about 50 nt, especiallyabout 100 nt, quite especially about 150 nt with 100% sequence identityto the corresponding part of the target nucleic acid. Preferably, forcalculating the sequence identity and designing the corresponding senseor antisense sequence, the number of gaps should be minimized,particularly for the shorter sense sequences.

dsRNA encoding chimeric genes according to the invention may comprise anintron, such as a heterologous intron, located e.g., in the spacersequence between the sense and antisense RNA regions in accordance withthe disclosure of WO 1999/53050 (incorporated herein by reference).

Synthetic Micro-RNAs (miRNAs).

The use of synthetic micro-RNAs to downregulate expression of aparticular gene in a plant cell, provides for very high sequencespecificity of the target gene, and thus allows conveniently todiscriminate between closely related alleles as target genes theexpression of which is to be downregulated.

Thus, in another embodiment of the invention, the biologically activeRNA or silencing RNA or inhibitory RNA molecule may be a microRNAmolecule, designed, synthesized and/or modulated to target and cause thecleavage ROD1 encoding gene or an orthologue thereof in a plant. Variousmethods have been described to generate and use miRNAs for a specifictarget gene (including but not limited to Schwab, et al. (2006, PlantCell, 18(5):1121-1133), WO 2006/044322, WO 2005/047505, EP 06009836, allincorporated herein by reference in their entirety, and particularly fortheir respective teachings relating to miRNA). Usually, an existingmiRNA scaffold is modified in the target gene recognizing portion sothat the generated miRNA now guides the RISC complex to cleave the RNAmolecules transcribed from the target nucleic acid. miRNA scaffoldscould be modified or synthesized such that the miRNA now comprises 21consecutive nucleotides of the ROD1 encoding nucleotide sequence or anorthologue thereof, such as the sequences represented in the Sequencelisting, and allowing mismatches according to the herein below describedrules.

Thus, in one embodiment, the invention provides a method for regulationof fatty acid unsaturation in seed oil comprising the steps of:

-   -   a. Introducing a chimeric gene into cells of an oilseed bearing        plant, said chimeric gene comprising the following operably        linked DNA regions:        -   i. a plant expressible promoter;        -   ii. a DNA region which upon introduction and transcription            in a plant cell is processed into a miRNA, whereby the miRNA            is capable of recognizing and guiding the cleavage of the            mRNA of a ROD1 encoding gene or an orthologue thereof of the            plant; and    -   iii. optionally, a 3′ DNA region involved in transcription        termination and polyadenylation.

The mentioned DNA region processed into a miRNA may comprise anucleotide sequence which is essentially complementary to a nucleotidesequence of at least 21 consecutive nucleotides of a ROD1 encoding geneor orthologue, provided that one or more of the following mismatches areallowed:

-   -   a. A mismatch between the nucleotide at the 5′ end of the miRNA        and the corresponding nucleotide sequence in the RNA molecule;    -   b. A mismatch between any one of the nucleotides in position 1        to position 9 of the miRNA and the corresponding nucleotide        sequence in the RNA molecule; and/or    -   c. Three mismatches between any one of the nucleotides in        position 12 to position 21 of the miRNA and the corresponding        nucleotide sequence in the RNA molecule provided that there are        no more than two consecutive mismatches.

As used herein, a “miRNA” is an RNA molecule of about 20 to 22nucleotides in length which can be loaded into a RISC complex and directthe cleavage of another RNA molecule, wherein the other RNA moleculecomprises a nucleotide sequence essentially complementary to thenucleotide sequence of the miRNA molecule whereby one or more of thefollowing mismatches may occur:

-   -   d. A mismatch between the nucleotide at the 5′ end of said miRNA        and the corresponding nucleotide sequence in the target RNA        molecule;    -   e. A mismatch between any one of the nucleotides in position 1        to position 9 of said miRNA and the corresponding nucleotide        sequence in the target RNA molecule;    -   f. Three mismatches between any one of the nucleotides in        position 12 to position 21 of said miRNA and the corresponding        nucleotide sequence in the target RNA molecule provided that        there are no more than two consecutive mismatches; and/or    -   g. No mismatch is allowed at positions 10 and 11 of the miRNA        (all miRNA positions are indicated starting from the 5′ end of        the miRNA molecule).

A miRNA is processed from a “pre-miRNA” molecule by proteins, such asDicerLike (DCL) proteins, present in any plant cell and loaded onto aRISC complex where it can guide the cleavage of the target RNAmolecules.

As used herein, a “pre-miRNA” molecule is an RNA molecule of about 100to about 200 nucleotides, preferably about 100 to about 130 nucleotideswhich can adopt a secondary structure comprising a double stranded RNAstem and a single stranded RNA loop and further comprising thenucleotide sequence of the miRNA (and its complement sequence) in thedouble stranded RNA stem. Preferably, the miRNA and its complement arelocated about 10 to about 20 nucleotides from the free ends of the miRNAdouble stranded RNA stem. The length and sequence of the single strandedloop region are not critical and may vary considerably, e.g., between 30and 50 nt in length. Preferably, the difference in free energy betweenunpaired and paired RNA structure is between −20 and −60 kcal/mole,particularly around −40 kcal/mole. The complementarity between the miRNAand the miRNA* need not be perfect and about 1 to 3 bulges of unpairednucleotides can be tolerated. The secondary structure adopted by an RNAmolecule can be predicted by computer algorithms conventional in the artsuch as mFOLD. The particular strand of the double stranded RNA stemfrom the pre-miRNA which is released by DCL activity and loaded onto theRISC complex is determined by the degree of complementarity at the 5′end, whereby the strand which at its 5′ end is the least involved inhydrogen bounding between the nucleotides of the different strands ofthe cleaved dsRNA stem is loaded onto the RISC complex and willdetermine the sequence specificity of the target RNA moleculedegradation. However, if empirically the miRNA molecule from aparticular synthetic pre-miRNA molecule is not functional (because the“wrong” strand is loaded on the RISC complex, it will be immediatelyevident that this problem can be solved by exchanging the position ofthe miRNA molecule and its complement on the respective strands of thedsRNA stem of the pre-miRNA molecule. As is known in the art, bindingbetween A and U involving two hydrogen bounds, or G and U involving twohydrogen bounds is less strong that between G and C involving threehydrogen bounds.

Naturally occurring miRNA molecules may be comprised within theirnaturally occurring pre-miRNA molecules but they can also be introducedinto existing pre-miRNA molecule scaffolds by exchanging the nucleotidesequence of the miRNA molecule normally processed from such existingpre-miRNA molecule for the nucleotide sequence of another miRNA ofinterest. The scaffold of the pre-miRNA can also be completelysynthetic. Likewise, synthetic miRNA molecules may be comprised within,and processed from, existing pre-miRNA molecule scaffolds or syntheticpre-miRNA scaffolds.

The pre-miRNA molecules (and consequently also the miRNA molecules) canbe conveniently introduced into a plant cell by providing the plantcells with a gene comprising a plant-expressible promoter operablylinked to a DNA region, which when transcribed yields the pre-miRNAmolecule. The plant expressible promoter may be the promoter naturallyassociated with the pre-miRNA molecule or it may be a heterologouspromoter.

Example 1 Materials and Methods

Plant Materials.

Mutant line rod1 in the Arabidopsis thaliana Col-0 background wasisolated from an M3 population of about 3,000 plants after mutagenesiswith ethyl methanesulfonate by directly analyzing the fatty acidcomposition of seed samples by gas chromatography (1). Plants were grownon soil in controlled environment chambers at 22° C. under continuousflorescent illumination (150 μmol quanta/m²/s).

Fatty Acid and Lipid Analysis.

The overall fatty acid compositions of seeds and other tissues weredetermined as described (2). Pulse-chase labeling was carried out indeveloping seeds harvested from siliques nine days after flowering. Theseeds were pulsed with [1-¹⁴C]glycerol or [1-¹⁴C]acetate for 15 min.After labeling, the tissues were chased with unlabelled acetate orglycerol. At different time intervals, total lipids were extracted andanalyzed in silica-TLC and the radioactivity in PC, DG and TG determinedby scintillation counting as described (3).

Genetic Analysis and Map-Based Cloning of ROD1.

To determine the genetic basis of the rod1 mutation, rod1 plants werecrossed to Col-0 wild-type (WT). F1 seeds showed a fatty acid profilesimilar to that of the WT parent. F1 plants were grown and allowed toself. Of 263 F2 plants analyzed, 69 had seed fatty acid profile similarto original rod1 seeds, while the remaining 194 had fatty acidcompositions similar to WT. This pattern of segregation is a good fit tothe hypothesized 3:1 ratio (χ²=0.21, p>0.05).

The rod1 locus was identified by map-based cloning using 800 F2 plantsderived from a cross between rod1 mutant and the Landsberg erecta WT.Initial screening by bulk segregant analysis of a set of 20 simplesequence length polymorphism (SSLP) markers that are evenly distributedin the Arabidopsis genome (4) resulted in the linkage of rod1 to themarker NGA162 in chromosome 3 (FIG. 2D). To fine map the rod1 locus, 196individual F2 plants were identified that were homozygous at the rod1locus indicated by increased 18:1 in seed fatty acid composition.Segregation analysis using available polymorphic SSLP markers vicinityof NGA162 delimited the rod1 mutation to an interval between NGA162 andNT204. More polymorphic markers were then designed using PCR primers,and subsequently located the rod1 locus in the region of chromosome 3covered by BAC clones MJK13, MQD17 and MSJ11 (FIG. 2D).

Within this region, eight genes were annotated as encoding proteins withknown or possible functions in lipid metabolism. After consideringpublished information (5, 6), Applicants amplified, by PCR, rod1 genomicDNA corresponding to six of the genes, including At3g15820. A G→Atransition was identified in this gene that is predicted to change Trp⁷⁶to a stop codon. The remaining five genes showed no changes from WT.

To confirm At3g15820 as the ROD1 locus, a PCR fragment of 3,961 bpcontaining the At3g15820 gene was amplified using genomic DNA extractedfrom Col-0 WT plants. This genomic fragment was cloned into a binaryvector pGate-Phas-DsRed at the AflII and EcoRI sites (2) and thentransferred into Agrobacterium tumefaciens strain GV3101 (pMP90) fortransformation of the rod1 mutant plants. Transformants were selectedbased on DsRed expression (7). Fatty acyl methyl esters derived fromindividual seeds of ten red transgenic seeds and three brownnon-transgenic seeds were used to determine seed fatty acid compositionusing gas chromatography.

ROD1 enzyme activity assays. The ROD1 open reading frame was amplifiedby PCR and cloned into the p424GPD yeast expression vector (8) forexpression in Saccharomyces cerevisiae under control of theglyceraldehydes-phosphate dehydrogenase promoter. The resultingconstruct p424ROD1 and the empty p424GPD vector were transformedseparately into the cells of HJ091 (cpt1::LEU2, ept1⁻) kindly providedby Dr. C. McMaster (Dalhousie University, NS, Canada). Expression ofROD1 transcripts was confirmed by RT-PCR (FIG. 7).

Yeast cells were inoculated from overnight cultures and grown to mid-logphase (OD₆₀₀=0.5-1.5) by rotary shaking at 30° C. in liquid syntheticminimal media lacking uracil and tryptophan supplemented with 2% glucose(Clontech, Mountain View, Calif.). To prepare microsomes, yeast cellswere harvested by centrifugation for 10 min at 1,000 g. The cell pelletwas washed once with sterile water and resuspended in ice-cold GTEbuffer (20% glycerol, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA) to prepare themembrane fraction using glass beads as described (9).CDP-Choline:diacylglycerol cholinephosphotransferase (CPT) assays(reactions in FIG. 9A) were conducted as described (9) using 0.1 moldiolein and 1 nmol [¹⁴C]CDP-choline as substrates.

The phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT)activities in membrane preparations of HJ091 cells transformed withp424GPD (mock) or p424ROD1 were determined as the amount of[¹⁴C]dioleoyl-PC produced from [¹⁴C-Glycerol]diolein (reaction A) or[¹⁴C-Choline]dimyristyl-phosphatidylcholine (reaction B). The substratesof 1.8 nmol (200,000 cpm) [¹⁴C-Glycerol]diolein (American RadiolabeledChemicals, St. Louis, Mo.) and 0.1 μmol dioleoyl-PC (reaction A) or 0.1μmol diolein and 1 μmol [¹⁴C-Choline]di-14:0-PC and 0.1 μmol dioleoyl-PC(reaction B) were dried under nitrogen gas and resuspended in 50 μl 4×reaction buffer (final concentrations: 50 mM MOPS/NaOH pH 7.5, 20 mMMgCl₂, 0.45% Triton X-100) with the aid of a sonicating bath (9).Reactions (200 μl) were started by adding 20-250 μg of microsomalproteins suspended in the GTE buffer. Unless otherwise indicated, assayswere incubated at 15° C. for 15 min and were terminated by the additionof 3 ml of chloroform/ethanol (2:1, v/v) followed by 1.5 ml of 0.9% KCl.Tubes were mixed by vortexing and phase separation was facilitated bycentrifugation at 2000 g for 2 min. The aqueous phase was aspirated andthe organic phase was washed twice with 1.5 ml of 40% (v/v) ethanol.Samples were analyzed by TLC on silica gel plates in a solvent system ofchloroform/methanol/water (65:25:4, by vol.) followed by phosphorimageranalysis or radioautography. Corresponding bands were scraped and radioactivities were determined by scintillation counting.

Expression of ROD1 and At3g15830.

Expression data for ROD1 (affymetrixarrayelement258249_s_at) database isshown in FIG. 8. The same array element also detects transcript of asecond gene, At3g15830, but data from the Arabidopsis Massively ParallelSignal Sequence (MPSS) database (mpss.udel.edu/at/) indicates that thissecond gene is only expressed in floral tissues (data not shown). Toconfirm these data, Applicants prepared RNA from germinating seedlings,rosette leaves, flowers and green siliques of WT plants, as well asgreen siliques of Rod1 mutant plants. Using oligonucleotide primersspecific for ROD1 and At3g15830, reverse-transcriptase PCR (RT-PCR) wasperformed on each of the RNA samples using the Superscript III one-stepsystem, according to the manufacturer's instructions (Invitrogen,Carlsbad, Calif.). The results shown in FIG. 7 indicate that expressionof At3g15830 is restricted to the flowers and that transcript of thisgene could not be detected in developing siliques of either WT or rod1plants. To test if At3g15830 also had a PDCT activity, a cDNA was clonedinto the p424GPD vector as described above. The resulting constructp424-At3g15830 was then transformed into HJ091 and its expression wasconfirmed by RT-PCR (FIG. 7, lane 16). PDCT assays using the samereaction conditions for ROD1 yielded no radiolabeled PC indicating thatthe At3g15830 protein does not have PDCT activity (data not shown).

Phylogenic Analyses.

The methods for producing the parsimony bootstrap tree (10) involved1000 bootstrap replicate data sets each analyzed using tree bisectionreconnection, steepest decent, and other settings to maximize thedetection of global optima or the maxmimization of the parsimonyoptimality criteria.

The methods for producing the Bayesian consensus tree (11) includedprior settings for the most complex WAG+F+I+G amino acid substitutionmodel and letting two Markov chains each run for 1,000,000 generation(sufficient for the two separate runs to converge before the secondparameter samples were made) while sampling every 10,000 generations atlikelihood stationarity in order to avoid autocorrelated parameterestimates.

REFERENCES CITED AND INCORPORATED HEREIN FOR THIS EXAMPLE 1

-   1. B. Lemieux, M. Miguel, C. Somerville, J. Browse, Theor. Appl.    Genetics 80, 234 (1990).-   2. C. Lu, M. Fulda, J. G. Wallis, J. Browse, Plant J. 45, 847    (2006).-   3. C. R. Slack, P. G. Roughan, N. Balasingham, Biochem. J. 170, 421    (1978).-   4. W. Lukowitz, C. S. Gillmor, W. R. Scheible, Plant Physiol. 123,    795 (2000).-   5. L. Fan, S. Zheng, X. Wang, Plant Cell 9, 2183 (1997).-   6. I. Heilmann, S. Mekhedov, B. King, J. Browse, J. Shanklin, Plant    Physiol. 136, 4237 (2004).-   7. A. R. Stuitje, Plant Biotech. J. 1, 301 (2003).-   8. D. Mumberg, R. Müller, M. Funk, Gene 156, 119 (1995).-   9. R. Hjelmstad, R. Bell, J. Biol. Chem. 266, 4357 (1991).-   10. D. L. Swofford. PAUP*. Phylogenetic Analysis Using Parsimony    (*and Other Methods), Version 4 (Sinauer Associates, Sunderland,    Mass., 2003).-   11. F. Ronquist, J. P. Huelsenbeck, Bioinformatics 19, 1572 (2003).

Example 2 The Arabidopsis Mutant Rod1 was Shown to have a MarkedDecrease in Polyunsaturated Fatty Acids in Seeds

Table 1A and 1B show that the Arabidopsis mutant rod1 of DH4 has amarked decrease in polyunsaturated fatty acids (PUFA) in seeds. Seedfatty acid compositions of the rod1 mutant differ from those of wildtype (WT) and the fad2 mutant of Arabidopsis thaliana. Compared to thefad2 mutant (5, 7), the fatty acid composition change in DH4 isrestricted to seed oil.

TABLES 1A and 1B Seed fatty acid compositions of the rod1 mutant differfrom those of wild type (WT) and the fad2 mutant of Arabidopsisthaliana. TAG, triacylglycerol; DAG, diacylglycerol; PC,phosphatidylcholine; PE, phosphatidylethanolamine Mol % of fatty acidspecies 16:0 18:0 18:1 18:2 18:3 20:1 A. Mature seeds TAG WT 8.4 3.115.1 29.2 19.9 18.6 rod1 8.5 3.3 32.8 13.8 15.6 20.6 fad2 6.0 2.4 65.00.2 1.6 24.0 WT × rod1 8.3 3.1 16.9 29.1 20.4 19.9 rod1 × fad2 8.3 2.420.1 24.3 21.0 22.4 B. Developing seeds at 9 days after flowering TAG WT9.2 3.7 17.9 30.5 16.2 18.6 rod1 9.9 3.8 39.1 14.2 12.3 17.9 DAG WT 17.06.5 18.2 33.8 14.2 4.3 rod1 16.1 5.3 33.8 22.2 10.9 8.6 PC WT 17.5 2.47.9 45.4 19.9 3.5 rod1 16.1 1.3 6.6 39.8 31.7 1.1 PE WT 30.6 3.3 7.535.4 18.9 1.3 rod1 33.2 3.2 6.4 34.9 18.3 1.6

As shown in Tables 1A and 1B, to further characterize the rod1 effect onseed lipid synthesis, fatty acid compositions of different classes ofglycerolipids were analyzed in mature (Table 1A) and developing seeds(Table 1B) at 9 days after flowering, the peak stage for fatty acidsynthesis. Compared to wild type, the seed oil of the rod1 mutant ofArabidopsis has a marked decrease in polyunsaturated fatty acids, butthere is no effect on the fatty acid compositions of leaf or roottissues. The rod1 mutant of Arabidopsis has an increased level of 18:1in both DAG and TAG, but displayed a decreased amount of 18:2 and 18:3(Tables 1A and 1B). However, for the fatty acids inphosphatidylethanolamine, little difference was detected between rod1and wild type. Interestingly, analysis of individual lipids fromdeveloping seeds of rod1 and wild type revealed that the rod1 mutant hada slightly decreased level of both 18:1 and 18:2 in PC compared to thewild type, and an increase in 18:3, relative to wild-type. Thus, thedeficiency in 18:2 and 18:3 was confined to the DAG and TAG ofdeveloping rod1 seeds. These findings are consistent with reducedtransfer of 18:1 into PC, and indicated that the reduced oleatedesaturation is not caused by desaturation activities, but due to areduced transfer of 18:1 into PC either via de novo synthesis fromdiacylglycerol (DAG) (8), or via the acyl-CoA:lyso-PC acyltransferaseexchange (9).

These changes are similar to, but smaller than, those observed in thefad2 mutants (5, 7) presenting the possibility that rod1 represented ahypomorphic allele of fad2. Whereas mutations at fad2 reduce PUFAsynthesis in leaves and roots as well as seeds, changes in fatty acidcomposition are only seen in seeds of rod1 plants (Table 1A and B, andTable 1C).

TABLE 1C Fatty acid composition in rod1 leaf and root lipids is similarto that of wild type Mol % of fatty acid species 16:0 16:3 18:0 18:118:2 18:3 Leaf WT 14.3 ± 0.4 13.7 ± 0.4 1.1 ± 0.1 3.8 ± 0.1 16.1 ± 0.546.3 ± 1.4 rod1 14.3 ± 0.3 14.8 ± 0.5 1.2 ± 0.1 3.8 ± 0.1 15.5 ± 0.444.9 ± 1.5 Root WT 22.9 ± 1.6 — 1.7 ± 0.3 7.6 ± 1.2 42.4 ± 1.5 25.7 ±1.6 rod1 23.6 ± 0.8 — 1.3 ± 0.1 6.8 ± 0.9 39.3 ± 0.8 29.0 ± 1.0

Crosses between rod1 and fad2 produced F1 seeds with PUFA levelsconsiderably higher than those of either parent, confirming that therod1 mutation is at a locus distinct from fad2. These and additionaltest crosses indicate that rod1 is a single, recessive Mendelianmutation.

As shown in Table 1A (mature seeds), genetic complementation testsbetween DH4 and the fad2 mutant further confirmed that the mutant locusrod1 in DH4 is not allelic to fad2. The mutation (as discussed in moredetail in EXAMPLE 4 herein below) occurred at the locus At3g15820 that,according to particular aspects of the present invention, normally(wild-type) encodes a novel phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) as determined by enzyme activity assayusing heterologous expression in yeast. Applicants have designated thismutant allele as rod1 (reduced oleate desaturation 1), which is a singlerecessive Mendelian mutation as determined by standard genetic analysis(data not shown).

Example 3

(The Arabidopsis mutant rod1 of DH4 was shown to have reduced oleatedesaturation in seed oil due to a reduced transfer of 18:1 into PC viade novo synthesis from diacylglycerol (DAG))

The growth, development and seed production of rod1 plants wereindistinguishable from WT. The weight of mature rod1 seeds wasindistinguishable from WT (17.7±0.2 and 17.9±0.1 μg/seed (av.±s.e.),respectively). Oil content of mature rod1 seeds was 4.9±0.32 μg/seed(av.±s.e.) compared with 4.6±0.19 μg/seed for WT, and the timing oflipid accumulation was similar in the two lines with a maximum 7-9 daysafter pollination (data not shown). The fatty acid compositions ofdifferent classes of glycerolipids extracted from seeds was analyzedduring this stage of maximum triglyceride synthesis. Compared to WT, therod1 mutant had substantially reduced levels of PUFAs in both TG and theimmediate precursor diglycerides (DG) (FIGS. 1E-1H). Surprisingly,however, PC contained increased PUFAs with the most highly unsaturatedfatty acid, 18:3, accounting for 31.7% of total acyl groups compared to19.9% in WT. The second most abundant phospholipid in seeds,phosphatidylethanolamine, does not have any major role in TG synthesis,and the fatty acid composition of this lipid was similar in the WT androd1 samples.

Because PC is the substrate for the FAD2 and FAD3 desaturases thatconvert 18:1 to 18:2 and 18:3 PUFAs, these data indicated thepossibility that the rod1 mutation reduces transfer of 18:1 into PC fordesaturation. Prior art models of TG synthesis in oilseeds propose that18:1 can enter the PC pool either by action of acyl-CoA:lyso-PCacyltransferase (LPCAT) or by the action of CDP-choline:DAGcholinephosphotransferase (CPT) on 18:1-DAG.

To distinguish whether the reduced oleate desaturation is due to areduced transfer of 18:1 into PC either via de novo synthesis fromdiacylglycerol (DAG) (8), or via the acyl-CoA:lyso-PC acyltransferaseexchange (9), developing seeds were labeled with radioactive acetate (tolabel fatty acids) (FIGS. 1C and 1D) and radioactive glycerol (to labelthe lipid backbone) (FIGS. 1A and 1B). In the glycerol chase experiment,PC was the most heavily labeled lipid (30%) in wild type seeds, and itremained relatively stable during the chasing period. A similar amountof label (27%) was also present in DAG at the end of pulse, whichdecreased during the chasing course, and consequently the lost label wasfound in TAG (FIG. 1A). In rod1 seeds, only 8% of label was detected inPC, but DAG contained 51% of total radioactivity at the end of pulse.The label present in TAG in rod1 seeds was at similar level to that inwild type. Similar results were also obtained from acetate chasingexperiments. These results indicate that rod1 seeds have reduced de novosynthesis of PC from DAG, since a lesion in acyl-CoA:lyso-PCacyltransferase would not be expected to restrict the flux of glycerolinto PC.

FIGS. 1A-1D show lipid synthesis in developing seeds of Arabidopsis.After 15 minutes of pulse with [14-C] labeled glycerol or acetate, thechase was carried out for 180 minutes. Radio activity in PC, DAG and TAGwere determined at 0, 30, 60 and 180 minute time points. Developingseeds were labeled with radioactive acetate (to label fatty acids)(FIGS. 1C and 1D) and radioactive glycerol (to label the lipid backbone)(FIGS. 1A and 1B).

Example 4

(Fine mapping of the Arabidopsis mutant rod1 of DH4 was performed andAt3g15820 was herein identified as the locus of the rod1 mutant, and forthe first time was shown not only to be aphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), butalso a PDCT that is highly expressed in developing seeds with thehighest level at stage 6 of seed development, which coincides the peakstage of storage deposition)

The de novo PC synthesis in oilseeds is known to be catalyzed by theCDP-choline:DAG cholinephosphotransferases (CPT). There are twohomologue genes for CPT in the Arabidopsis genome (10). Applicants,therefore, initially presumed (incorrectly) that rod1 was likely amutation in one of the two Arabidopsis CDP-choline:DAGcholinephosphotransferase genes, At1g13560 (AAPT1) or At3g25585 (AAPT2).Surprisingly, however, Applicants' initial mapping data for rod1 placedthis gene approximately 20 cM north of AAPT2 on chromosome 3.

Specifically, the genomic DNA covering the two CPT coding regions wassequenced, but there were no sequence mutations in the CPT genes in therod1 mutant. This result indicated that the CPTs in rod1 functionnormally and that there was likely another mechanism(s) for synthesizingPC in developing seeds. A map-based cloning approach was thereforeconducted to identify the rod1 locus using F2 plants derived from across between rod1 and the Landsberg erecta wild type (see “Geneticanalysis and map-based cloning of ROD1” under “Materials and Methods” ofExample 1 above). This approach allowed identification of a mutation atthe locus At3g15820 on chromosome 3. A single nucleotide change from Gto A in the first exon of At3g15820 resulted in a change of Trp⁷⁶ to astop codon (see FIG. 2C). Compared to the wild-type nucleic acidsequence (SEQ ID NO:2), the rod1 allele nucleic acid sequence (SEQ IDNO:4) of this gene coding sequence contains a mutation that creates astop codon at residue 76 of the predicted opening reading frame.

The identity of ROD1 and At3g15820 was subsequently confirmed bycomplementing the rod1 mutant with a wild-type ˜4-kb genomic sequence(SEQ ID NO:1) including the At3g15820 coding regions and its endogenouspromoter and terminator (4 kb genomic fragment of wild-type DNA,including the coding region of At3g15820 and a total of 2 kb of 5′ and3′ flanking sequence) (FIG. 2A). This DNA fragment was cloned into abinary plant transformation vector using the DsRed as a selection marker(11). Transgenic seeds were identified by DsRed expression, and theirfatty acyl methyl esters (FAMEs) were analyzed by gas chromatography andcompared with those of untransformed seeds from the same plants. Thefatty acid composition of the transgenic seeds was nearly identical tothat of the wild type (FIG. 2B), confirming that the rod1 mutation isindeed at the At3g15820 locus.

FIGS. 2A-2C show that the ROD1 gene was identified as At3g15820 inArabidopsis. (A) The structure of the ROD1 gene with the position of themolecular lesion in the mutant. An approximately 4 Kb region (SEQ IDNO:1) showing exons (bold arrows) and untranslated regions (boxes) wasused to complement mutation in rod1. (B) Comparison of seed fatty acidcompositions of the At3g15820-transformants (white hatched) and the Colwild-type (white) indicated that At3g15820 fully restored the rod1mutation (black), thus confirming the identity of ROD1. (C) Deducedamino acid sequence (SEQ ID NO:3) of At3g15820 arranged to show putativetransmembrane regions predicted by HMMTOP (underlined). The asteriskmarks the position of the change of the codon for Trp 76 into a stopcodon) in the mutant sequence SEQ ID NO:4 (single point mutation; G to Ain the first exon sequence). The putative lipid phosphate phosphatasemotif is shown in bold and italics.

FIG. 4 shows the ROD1 mutant truncated amino acid sequence (SEQ ID NO:5)in DH4. According to particular aspects, aphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)mutant (rod1) coding sequence (SEQ ID NO:4) comprises a G>A change atnucleotide position 228, resulting in premature termination of the PDCTprotein to provide a 75 amino acid truncated ROD1 mutant sequence (SEQID NO:5).

Analyzing publicly available microarray data(https://www.genevestigator.ethz.ch/) (12) indicated that At3g15820 ishighly expressed in developing seeds with the highest level at stage 6of seed development, which coincides the peak stage of storagedeposition. This is in agreement with the seed-specific decreased oleatedesaturation in the rod1 mutant (Table 1). According to additionalaspects, the only gene in Arabidopsis that shares high homology toAt3g15820 is At3g15830, which is located just 1.2 kb downstream.However, At3g15830 is only expressed in inflorescence, and Applicants'RT-PCR results also confirmed that At3g15830 is not expressed indeveloping seeds.

Significantly, At3g15820 was annotated as a putative type 2 phosphatidicacid phosphatase (PAP2)-like protein, however, upon analysis byApplicants, it did not show strong homology to known characterized PAPgenes in Arabidopsis (AtLPP1, AtLPP2 and AtLPP3) (13, 14). Specifically,ROD1 was rechecked against the PFAM database, and the E-value foridentification of a PAP2 domain (0.017) was above the recommended cutoffNot only was the sequence match poor, with only 8 of the 15 mostconserved residues being present in ROD1, but the alignment also showedthat ROD1 had (would need to have) deletions totaling 61 residues (outof 176) in the center of the motif sequence. The Arabidopsis genomecontains at least four genes with clearly identified PAP2 domains (Evalues <e⁻⁴⁰) including LPP1 (At3g02600) and LPP2 (At1g15080), whichhave been shown to have PA phosphatase activity. Applicants determined,therefore, that ROD1 contains essentially no sequence homology to thesetrue PAP2 orthologues, and thus concluded that ROD1 encodes a differentfunction. Additionally, when expressed in yeast (Saccharomycescerevisiae) by Applicants, ROD1 did not confer significantly higher PAPactivity than the control strain. These results indicated that ROD1 wasno likely to possess PA phosphatase activity.

The position-specific iterated BLAST (PSI-BLAST) algorithm was used, andthe third iteration identified a non-plant protein phosphatidylcholine:ceramide cholinephosphotransferase (a mammalianphosphatidylcholine:ceramide cholinephosphotransferase (EC 2.7.8.27)),which belongs to a large family of lipid phosphate phosphatases (LPP).This enzyme, also called sphingomyelin synthase (15), catalyzes thetransfer of the phosphocholine head group from PC to the alcohol groupof ceramide. Applicants appreciated that in the structures andmetabolism of sphingolipids, ceramide has a role that is analogous toDAG for glycerolipids. ROD1 is a membrane bound protein containing 5predicted transmembrane domains according to the program HMMTOP (16),and its sequence contains a LPP motif (FIG. 2C). These results suggestedto Applicants that ROD1 would be able to transfer the phosphocholinehead group from PC to DAG in plants, an analogous reaction of PC withceramide in animals. Applicants, therefore, termed this putative newenzyme as phosphatidylcholine: diacylglycerol cholinephosphotransferase(PDCT).

Phylogenetic analysis places ROD1 in close relationship to the SMS1 andSMS2 proteins within the LPT family (FIG. 5) and topology predictionprograms identify ROD1 as an integral-membrane protein with up to sixputative transmembrane domains—similar to predictions for other LPTproteins. In addition, five highly conserved residues in the C2 and C3domains of SMS1, SMS2 and other LPT proteins are identified atcomparable positions in the ROD1 protein (FIG. 2C, and FIG. 5). Plantsdo not contain sphingomyelin, but the structure of ceramide is similarto that of DG so Applicants considered the possibility that ROD1catalyzes transfer of phosphocholine from PC to DG in a reactionanalogous to that mediated by SMS in animals. Following biochemicalconvention, Applicants designate this putative enzyme asphosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) inthe IUPAC subclass EC 2.7.8.

Testing ROD1 for PDCT Activity.

CDP-choline:DAG cholinephosphotransferase is responsible for the initialsynthesis of PC and the observation that this reaction is readilyreversible in vitro has been invoked to explain the rapid equilibrationbetween the PC and DAG pools that occurs in developing oilseeds.Experimentally (in labeling studies or assays using membranepreparations), however, it is difficult to distinguish between thedouble action of CDP-choline:DAG cholinephosphotransferase (reactionscheme 1 below) and the single-step reaction catalyzed by PC:DAGcholinephosphotransferase (reaction scheme 2 below). Reaction scheme 2is simple and analogous to the reaction of PC with ceramide, but toApplicants' knowledge, has not previously been described in anyorganism—perhaps largely due to the difficulty of distinguishing it fromreaction scheme 1. Nonetheless, analogous transfer reactions are known.

Therefore, Applicants reasoned that the most straightforward way to testROD1 for PC:DAG cholinephosphotransferase activity would be to expressthe recombinant protein in a double-mutant yeast strain (e.g., HJ091)lacking all CDP-choline:DAG cholinephosphotransferase activity. In thisstrain, PC is only synthesized by a reaction sequence involvingdecarboxylation of phosphatidylserine (PS) to phosphatidylethanolaine(PE) followed by three cycles of methylation to produce PC. Microsomalpreparations from this yeast strain will not support incorporation of[³H]-labeled DAG or [¹⁴C]-labeled choline (supplied as CDP-choline) intoPC. Applicants, therefore reasoned that if expression of ROD1 results inconversion of ³H-DAG to PC, it will indicate that ROD1 acts as a PC:DAGcholine-phosphotransferase.

Therefore, to test ROD1 for PDCT activity, the cDNA of At3g15820 wasexpressed under control of the inducible GAL1 promoter in a Δcpt Δeptdouble-mutant yeast strain HJ091 (17) lacking allCDP-choline:diacylglycerol cholinephosphotransferase activities. In thisstrain, PC is only synthesized by a reaction sequence involvingdecarboxylation of PS to PE, followed by three cycles of methylationreactions (17). Microsomal preparations from this yeast strain do notsupport incorporation of diacylglycerol and CDP-choline into PC. Asexpected, the ROD1-transformed yeast microsomes are not able tosynthesize radioactive PC when incubated with diolein and [14C]-labeledCDP-Choline (not shown), or [¹⁴C-glycerol]-diolein and CDP-choline (FIG.3A). However, [¹⁴C]-labeled PC was clearly detected when [¹⁴C]-dioleinand PC were provided as substrates. Since the [¹⁴C]-radiolabel was onthe glycerol backbone of diolein, the radioactive PC was apparentlyresulted from the phosphocholine head group transfer into diolein.

To further confirm this PDCT activity, the ROD1-transformed yeastmicrosomes were incubated with [¹⁴C-Choline]-PC and di-8:0-DAG. As shownin FIG. 3B, radiolabeled di-8:0-PC was detected in this assay,indicating the transfer of the phosphocholine headgroup to thedi-8:0-DAG. These results indicate that ROD1 does not possess PAphosphatase activity, and substantially confirms that ROD1 ratherconfers a PDCT activity, which is consistent with the fact that the rod1mutant is defective in PC synthesis in developing seeds.

FIGS. 3A-3C show that ROD1 functions as aphosphatidylcholine:diacylglycerol cholinephosphotransferase. Microsomesfrom yeast strain HJ091 transformed with ROD1 convert[14C]glycerol-labeled dioleoylglycerol into1,2-dioleoyl-sn-glycero-3-phosphocholine (A), or yields1,2-dioctanoyl-sn-glycero-3-phosphocholine when incubated with[¹⁴C]Choline-labeled dipalmitoyl phosphocholine and1,2-dioctanoyl-sn-3-glycerol (B). No new radio-labeled PC products weredetected in reactions using HJ091 transformed with the empty vectorpYES2 (A, B, C).

More specifically, Microsomal preparations from JH091 cells expressingROD1 and from empty-vector controls were first tested for the ability tosynthesize PC from DG and CDP-[¹⁴C]choline (20). No activity wasdetected in either control microsomes or those from cells expressingROD1 (FIG. 9A). However, ¹⁴C-labeled PC was produced when ROD1microsomes were incubated with dioleoyl-[¹⁴C]glycerol and this activitywas enhanced in the presence of added PC (FIG. 9B). Control microsomesdid not have activity in this assay and ROD1 microsomes that had beenboiled prior to assay were also inactive. Because the [¹⁴C] radiolabelwas in the glycerol moiety of the [¹⁴C]-DG substrate, these assaysindicate that ROD1 synthesizes [¹⁴C]-PC PC by transfer of thephosphocholine headgroup from PC to [¹⁴C]-DG. The activity observed inassays without added PC presumably relied on endogenous PC of the yeastmicrosomes.

Assays with other possible phosphocholine donors indicated that only thephosphocholine headgroup of PC was accessible to the ROD1 enzyme.Addition of 1 mM CDP-choline, phosphocholine or lyso-PC did not support[¹⁴C]-PC synthesis at rates higher than ROD1 microsomes without added PC(FIG. 9C). To specifically test for transfer of the PC headgroup, weincubated microsomes with [¹⁴C]choline-labeled dimyristoyl-PC andunlabeled dioleoyl-DG. In this assay, ROD1 microsomes, but not thecontrol, synthesized dioleoyl-[¹⁴C]-PC, which separates from thedimyristoyl-[¹⁴C]PC substrate on thin layer chromatography (FIG. 9D).Additional assays indicated that the PDCT activity was highest at pH6.5-7 (FIG. 9E). Under the assay conditions used, [¹⁴C]-PC synthesisincreased linearly with incubation time up to 3 min, and with proteinconcentration up to 50 μg of microsomal protein (FIG. 9F, G). Underoptimized assay conditions, PDCT activity was 0.6 nmol/min/mg microsomalprotein in the absence of added PC, increasing to 4.5 nmol/min/mg with 1mM added PC (FIG. 9H).

In summary, Applicants' analyses of the rod1 mutant in Arabidopsis andbiochemical functional assays using heterologous yeast expressionsurprisingly establishes that ROD1 (At3g15820) functions as aphosphatidylcholine diacylglycerol cholinephosphotransferase.Specifically, it is responsible for the flux from DAG into PC indeveloping seeds of seed oil plants, including but not limited to, e.g.,Arabidopsis).

A minimum estimate of the flux through PDCT can be made from the data inTable 1. In WT seeds, 49.1% of fatty acids are 18:2+18:3, the twoproducts of FAD2 desaturation on PC. In the rod1 mutant, these fattyacids are only 29.4% of the total, indicating that 40%

$\frac{49.1 - 29.4}{49.1} \times \frac{100}{1}$of the 18:1 converted to 18:2+18:3 enters PC via the PDCT enzyme.Sequences homologous to Arabidopsis ROD1 are identifiable in many higherplants, including oil crops such as canola (Brassica napus), sunflower(Helianthus annua) and castor bean (Ricinus communis) and it is thuslikely that PDCT is an important enzyme of TG synthesis in many plants.

Our discovery of PDCT in Arabidopsis has important implication forunderstanding TG synthesis and for using biotechnology to modify thefatty acid composition of plant oils. For example, because PDCTcontributes to the control of PUFA synthesis in seeds, regulation ofROD1 expression could reduce the need for hydrogenation of oils, and theattendant production of unhealthy trans fats (3, 4), as well asproviding for the production of biofuels with increased oxidativestability (22). Because PC is also the substrate for enzymes thatproduce hydroxy-, epoxy-, acetylenic and other modified fatty acids(23-25), our discovery of PDCT provides many opportunities to betterunderstand TG synthesis in different oilseed species and to improve thefatty-acid profiles of vegetable oils for both human health andindustrial applications.

Example 5 ROD1 (At3g15820) Orthologs were Identified that haveSignificant Sequence Homology/Identity

According to further aspects of the present invention, many major fattyacid modifications in nature are accomplished by acting on fatty acylchains esterified on PC, and orthologs of ROD1 genes were hereinidentified in many other plant species including oil crops such asrapeseed and castor (Ricinus communis), etc.

According to particular aspects, expression of the cDNAs in yeast strainHJ091 provides for activity assays of the encoded protein as we did forROD1. Once the canola ROD1orthologue(s) have been identified, they canbe targeted for down-regulation and the resulting plants evaluated fortheir value in breeding programs to produce lines with increased 18:1 inthe seed oil.

Tables 2 and 3 show nucleotide similarity (% identity) and proteinsequence similarity (% identity), respectively, for exemplary ROD1orthologs from Brassica (SEQ ID NO:6; SEQ ID NO:7), Moss (SEQ ID NO:16;SEQ ID NO:17), Spruce (SEQ ID NO:14; SEQ ID NO:15), Grape (SEQ ID NO:12;SEQ ID NO:13), Rice (SEQ ID NO:10; SEQ ID NO:11) and Castor (SEQ IDNO:8; SEQ ID NO:9), showing a range of nucleic acid identity from about46 to 80%, and range of protein sequence identity from about 42 to 85%.According to additional aspects, cloned cDNAs encoding for theseorthologs transgenically complement the Arabidopsis rod1 mutant.

TABLE 2 ROD1 nucleotide similarity (% identical) Castor (SEQ ID BrassicaMoss Spruce Grape Rice NO: 8) Arabidopsis 80 55 54 66 55 62 (SEQ ID NO:18) Brassica 52 52 64 53 60 (SEQ ID NO: 6) Moss 59 51 46 52 (SEQ ID NO:16) Sitka spruce 61 47 56 (SEQ ID NO: 14) Wine Grape 64 71 (SEQ ID NO:12) Rice 54 (SEQ ID NO: 10)

TABLE 3 ROD1 protein sequence similarity (% identical) Grape (SEQ IDBrassica Moss Rice Spruce Castor NO: 13) Arabidopsis 85 48 46 58 64 71(SEQ ID NO: 3) Brassica 46 48 59 64 72 (SEQ ID NO: 7) Moss 42 48 45 47(SEQ ID NO: 17) Rice 46 51 56 (SEQ ID NO: 11) Sitka spruce 58 65 (SEQ IDNO: 15) Castor 72 (SEQ ID NO: 9)

Therefore, according to preferred embodiments, manipulation of PDCT inoilseeds provides a novel approach (e.g., genetic approach) to modifyfatty acid profiles of plant oils to customize and/or optimize plantoils in view of particular end-use requirements. Regardless of theenzymatic identity of ROD1, the effects of the rod1 mutation on thefatty acid compositions of TAG, DAG and PC indicate that down-regulationof ROD1 homologues in crop plants has substantial utility to modulate orreduce levels of 18:2 and 18:3 in the oil while maintaining unsaturationof membrane lipids.

Example 6 Brassica napus, Brassica rapa (2038 and 370) and Brassicaoleracea Sequences

According to further aspects of the present invention, the Brassicanapus unigene Bna.6194 is identified as the true Arabidopsis ROD1(At3g15820) homologue. Applicants named Bna.6194 as BnROD1. QuantitativeRT-PCR showed that BnROD1 is highly expressed in canola developingseeds. Brassica napus is an amphidipoid including Brassica rapa andBrassica oleracea two subgenomes.

The sequence alignment also suggested that BnROD1 might be the truehomologue of Brassica rapa unigene Bra. 2038 and Brassica oleraceaES948687. Although another Brassica rapa unigene Bra.370 also shareshighly identity with BnROD1, it cannot be amplified by RT-PCR fromdeveloping seed cDNA.

Unigene Bna.6194 (or TC71619 in TIGR) (SEQ ID NO: 27)GAGATGAGAAAATAGCAAAGACTTGCGTAAACGTCGCTCTCAAACCTCATCTCATACTCATCGTTTTCGTATGAGTTTTTGTAGCCCAAACAATCTTCCTTTCTACAGTTTATAATATAAGAAACAATACTTCCTTCGTAATCTCCGCCTCGTATCTCTTATATAACTCATCTCTCTAAACCTAAAAAATGTTCCTCTCCGTTAAATCTAACGGTCATGTCAACTAATACCGTCGTCCCTCTCCGTCGCAGATCTAACGGATATCACACTAACGGCGTGGCCTTTAACGGAATGGATAATATTGTCAAGAAAACCGACGACTGCTACACCAACGGCAACGGCAACGGAGGAGTAGAGAGAAGCAAAGCCTCGTTTCTGACATGGACCATGCGTGACGCTGTCTACGTAGCGAGATACCATTGGATACCGTGTTTCTTTGCGGTCGGAGTTCTGTTCTTTATGGGGGTTGAGTACACGCTCCAGATGGTTCCGGCGAAGTCTGAGCCGTTCGATATTGGGTTTGTGGCCACGCGCTCTCTAAACCGCGTCTTGGCGAGTTCACCGGATCTTAACACCCTTTTAGCGGCTCTAAACACGGTATTCGTAGCGATGCAAACGACGTATATTGTATGGACATGGTTGATGGAAGGAAGACCACGAGCCACTATCTCGGCTTGCTTCATGTTTACTTGTCGCGGCATTCTTGGTTACTCTACTCAGCTCCCTCTACCACAGGATTTTTTAGGATCAGGAGTTGATTTTCCGGTGGGAAACGTCTCATTCTTCCTCTTCTATTCTGGCCACGTAGCCGGTTCAATGATCGCATCCTTGGACATGAGGAGAATGCAGAGGTTGAGACTAGCGATGCTTTTTGACATCCTCAACATATTACAATCGATCAGACTGCTCGGGACGAGAGGACACTACACGATCGATCTTGCGGTCGGAGTTGGCGCTGGGATTCTCTTTGACTCATTGGCCGGGAAGTACGAAGAGATGATGAGCAAGAGACACAATTTAGCCAATGGTTTTAGTTTGATTTCTAAAGACTCGCTAGTCAATTAATCTTTTGTTTTCATTTTAAATGATTAGTTGAACTTGAACATATTTGATTTAGTTAAAGTCCAATGAATTACAunderlined areas are the primers used for amplification of BnROD1 ORF.Bold-face areas the primers used for real-time PCR.BnROD1 coding region sequence (from Bna6194) (SEQ ID NO: 19)   1ATGTCAACTA ATACCGTCGT CCCTCTCCGT CGCAGATCTA ACGGATATCA CACTAACGGC  61GTGGCCTTTA ACGGAATGGA TAATATTGTC AAGAAAACCG ACGACTGCTA CACCAACGGC 121AACGGCAACG GAGGAGTAGA GAGAAGCAAA GCCTCGTTTC TGACATGGAC CATGCGTGAC 181GCTGTCTACG TAGCGAGATA CCATTGGATA CCGTGTTTCT TTGCGGTCGG AGTTCTGTTC 241TTTATGGGGG TTGAGTACAC GCTCCAGATG GTTCCGGCGA AGTCTGAGCC GTTCGATATT 301GGGTTTGTGG CCACGCGCTC TCTAAACCGC GTCTTGGCGA GTTCACCGGA TCTTAACACC 361CTTTTAGCGG CTCTAAACAC GGTATTCGTA GCGATGCAAA CGACGTATAT TGTATGGACA 421TGGTTGATGG AAGGAAGACC ACGAGCCACT ATCTCGGCTT GCTTCATGTT TACTTGTCGC 481GGCATTCTTG GTTACTCTAC TCAGCTCCCT CTACCACAGG ATTTTTTAGG ATCAGGAGTT 541GATTTTCCGG TGGGAAACGT CTCATTCTTC CTCTTCTATT CTGGCCACGT AGCCGGTTCA 601ATGATCGCAT CCTTGGACAT GAGGAGAATG CAGAGGTTGA GACTAGCGAT GCTTTTTGAC 661ATCCTCAACA TATTACAATC GATCAGACTG CTCGGGACGA GAGGACACTA CACGATCGAT 721CTTGCGGTCG GAGTTGGCGC TGGGATTCTC TTTGACTCAT TGGCCGGGAA GTACGAAGAG 781ATGATGAGCA AGAGACACAA TTTAGCCAAT GGTTTTAGTT TGATTTCTAA AGACTCGCTA 841GTCAATTAA BnROD1 translated ORF sequence ) (SEQ ID NO: 20)MSTNTVVPLRRRSNGYHTNGVAFNGMDNIVKKTDDCYTNGNGNGGVERSKASFLTWTMRDAVYVARYHWIPCFFAVGVLFFMGVEYTLQMVPAKSEPFDIGFVATRSLNRVLASSPDLNTLLAALNTVFVAMQTTYIVWTWLMEGRPRATISACFMFTCRGILGYSTQLPLPQDFLGSGVDFPVGNVSFFLFYSGHVAGSMIASLDMRRMQRLRLAMLFDILNILQSIRLLGTRGHYTIDLAVGVGAGILFDSLAGKYEEMMSKRHNLANGFSLISKDSLVN Unigene Bra. 2038 (Brassica rapa) )(SEQ ID NO: 28)GATGGTAAGGAAACTCTCGTACTCTTCTCTATCTTTTTGTGTGTGTTTCTCGTGTAAAATATTATACACTTAAGACGTATAAAAAGAACAACAAGTAAAGCCCAACAAAGACAGATGAGAAAATAGCAAAGACTTGCGTAAACGTCGCTCTCAAACCTCATCTCATACTCATCGTTTTCGTATGAGTTTTTGTAGCCCAAACAATCTTCCTTTCTACAGTTTATAATATAAGAAACAATACTTCCTTCGTAATCTCCGCCTCGTATCTCTTATATAACTCATCTCTCTAAACCTAAAAAATGTTCCTCTCCGTTAAATCTAACGGTCATGTCAACTAATACCGTCGTCCCTCTCCGTCGCAGATCTAACGGATATCACACTAACGGCGTGGCCTTTAACGGAATGGAGAACATTGTCAAGAAAACCGACGACTGCTACACCAACGGCAACGGCAACGGAGGAGTAGAGAGAAGCAAAGCCTCGTTTCTGACATGGACCATGCGTGACGCTGTCTACGTAGCGAGATACCATTGGATACCGTGTTTCTTTGCGGTCGGAGTTCTGTTCTTTATGGGGGTTGAGTACACGCTCCAGATGGTTCCGGCGAAGTCTGAGCCGTTCGATATTGGGTTTGTGGCCACGCGCTCTCTGAACCGCGTCTTGGCGAGTTCACCGGATCTTAACACCCTTTTAGCGGCTCTAAACACGGTATTCGTAGCGATGCAGACGACGTATATTGTATGGACATGGTTGATGGAAGGAAGACCACGAGCCACTATCTCGGCTTGCTTCATGTTTACTTGTCGCGGCATTCTTGGTTACTCTACTCAGCTCCCTCTACCACAGGATTTTTTAGGATCAGGAGTTGATTTTCCGGTGGGAAACGTCTCATTCTTCCTCTTCTATTCTGGCCACGTAGCCGGTTCAATGATCGCATCCTTGGACATGAGGAGAATGCAGAGGTTGAGACTAGCGATGCTTTTTGACATCCTCAACATATTACAATCGATCAGACTGCTCGGGACGAGAGGACACTACACGATCGATCTTGCGGTCGGAGTTGGCGCTGGGATTCTCTTTGACTCATTGGCCGGGAAGTACGAAGAGATGATGAGCAAGAGACACAATTTAGCCAATGGTTTTAGTTTGATTTCTAAAGACTCGCTAGTCAATTAATCTTTTGTTTTTATTTTAAATGATTAGTTGAACTTGAACATATTTGATTTAGTTAAAGTCCAATGAATTACATTTTTTTCTTTCAACTTTAATTGAATAGGGTTTCATTAGTTTACTTGAACCTAATTAAATGTGTACGTTATTGTGAAATAAAGAAGTTTGTTGTGGCCTTCCTACAACTATTTCATCAAAAAAAAAAAAAA BrROD1 Coding sequence: )(SEQ ID NO: 21)ATGTCAACTAATACCGTCGTCCCTCTCCGTCGCAGATCTAACGGATATCACACTAACGGCGTGGCCTTTAACGGAATGGAGAACATTGTCAAGAAAACCGACGACTGCTACACCAACGGCAACGGCAACGGAGGAGTAGAGAGAAGCAAAGCCTCGTTTCTGACATGGACCATGCGTGACGCTGTCTACGTAGCGAGATACCATTGGATACCGTGTTTCTTTGCGGTCGGAGTTCTGTTCTTTATGGGGGTTGAGTACACGCTCCAGATGGTTCCGGCGAAGTCTGAGCCGTTCGATATTGGGTTTGTGGCCACGCGCTCTCTGAACCGCGTCTTGGCGAGTTCACCGGATCTTAACACCCTTTTAGCGGCTCTAAACACGGTATTCGTAGCGATGCAGACGACGTATATTGTATGGACATGGTTGATGGAAGGAAGACCACGAGCCACTATCTCGGCTTGCTTCATGTTTACTTGTCGCGGCATTCTTGGTTACTCTACTCAGCTCCCTCTACCACAGGATTTTTTAGGATCAGGAGTTGATTTTCCGGTGGGAAACGTCTCATTCTTCCTCTTCTATTCTGGCCACGTAGCCGGTTCAATGATCGCATCCTTGGACATGAGGAGAATGCAGAGGTTGAGACTAGCGATGCTTTTTGACATCCTCAACATATTACAATCGATCAGACTGCTCGGGACGAGAGGACACTACACGATCGATCTTGCGGTCGGAGTTGGCGCTGGGATTCTCTTTGACTCATTGGCCGGGAAGTACGAAGAGATGATGAGCAAGAGACACAATTTAGCCAATGGTTTTAGTTTGATTTCTAAAGACTCGCTAGTCAATTAA Unigene Bra. 2038 translated ORF sequence(SEQ ID NO: 22)MSTNTVVPLRRRSNGYHTNGVAFNGMENIVKKTDDCYTNGNGNGGVERSKASFLTWTMRDAVYVARYHWIPCFFAVGVLFFMGVEYTLQMVPAKSEPFDIGFVATRSLNRVLASSPDLNTLLAALNTVFVAMQTTYIVWTWLMEGRPRATISACFMFTCRGILGYSTQLPLPQDFLGSGVDFPVGNVSFFLFYSGHVAGSMIASLDMRRMQRLRLAMLFDILNILQSIRLLGTRGHYTIDLAVGVGAGILFDSLAGKYEEMMSKRHNLANGFSLISKDSLVN ES948687 (Brassica oleracea) )(SEQ ID NO: 29)GAGATGAGAAAATAGCAAAGACTTGCGTAAACGTCGCTCTCAAATCTCATCTCATACTCATCGTTTTCGTATGAGTTTTTGTAGCCCAAACAATCTTCCTTTCTACGGTTTATAATATAAGAAACAATACTTCCTTCGTAATCTCCGCCTTGTATCTCTTATATAACTCATCTCTCTAAACCTAAAAAATGTTCCTCTCCGTTAAATCTAACGGTCATGTCAACTAATACCGTCGTCCCTCTCCGTCGCAGATCTAACGGATATCACACTAACGGCGTGGCCTTCAACGGAATGGAGAACATTGTCAAGAAAACCGACGACTGCTACACCAATGGCAACGGAGTAGGAGGGAAGAGCAAGGCGTCATTTCTGACATGGACCATGCGTGACGCTGTCTTCGTAGCGAGATACCATTGGATACCATGTTTCTTTGCTGTCGGAGTTCTGTTCTTTATGGGGGTTGAGTACACGCTCCAGATGGTTCCGGCGAAGTCTGAGCCGTTCGATATTGGGTTTGTGGCCACGCGCTCTCTGAACCGCGTCTTGGCGAGTTCACCGGATCTTAACACCCTTTTAGCGGCTCTAAACACGGTATTCGTAGCGATGCAAACGACGTATATTG ...ES948687 Partial coding sequence: (SEQ ID NO: 23)ATGTCAACTAATACCGTCGTCCCTCTCCGTCGCAGATCTAACGGATATCACACTAACGGCGTGGCCTTCAACGGAATGGAGAACATTGTCAAGAAAACCGACGACTGCTACACCAATGGCAACGGAGTAGGAGGGAAGAGCAAGGCGTCATTTCTGACATGGACCATGCGTGACGCTGTCTTCGTAGCGAGATACCATTGGATACCATGTTTCTTTGCTGTCGGAGTTCTGTTCTTTATGGGGGTTGAGTACACGCTCCAGATGGTTCCGGCGAAGTCTGAGCCGTTCGATATTGGGTTTGTGGCCACGCGCTCTCTGAACCGCGTCTTGGCGAGTTCACCGGATCTTAACACCCTTTTAGCGGCTCTAAACACGGTATTCGTAGCGATGCAAACGACGTATATTG ... ES948687 translated amino acid sequence )(SEQ ID NO: 24)MSTNTVVPLRRRSNGYHTNGVAFNGMENIVKKTDDCYTNGNGVGGKSKASFLTWTMRDAVFVARYHWIPCFFAVGVLFFMGVEYTLQMVPAKSEPFDIGFVATRSLNRVLASSPDLNTLLAALNTVFVAMQTTYI ... Unigene Bra. 370 (Brassica rapa) ) (SEQ ID NO: 30)GCTCTCAAATCTCATATTCATCGTTTTCGTATGAACTTTTGTAGCCCAAACAACCTTCCTTTCCTTCCACAAGTTTCATATAATATCTCTTATATAACCCATCTCTCTAAGCCTCTCAAAACGTTCTTCTCCGTTAAATCTAACGGCCATGTCAACTACAACAATCGTCCCTCTCCGTCGCACTTCTAACTCTCTCAATGAATACCACACTAACGCAGTCGCCTTTGACGGAATCGTCGGGTCAGCAAGTACTAGCCAAATGGAGGAGATTGTTACGCAAACCGACGACTGCTACGCCAACCCCAACGGAGATGGAGGGAGAAGCAAGACGTCGTTAATGACGTGGAGGATGTGCAATCCTGTCCACGTGGTGAGAGTCCATTGGATACCGTGTTTGTTTGCGGTAGGAGTTCTGTTCTTCACGTGCGTAGAGGAGTACATGCTCCAGATGATTCCGGCGAGTTCTGAGCCGTTCGATATTGGTTTTGTGGCGACGGGCTCTCTGTATCGCCTCTTGGCTTCTTCACCGGATCTTAATACCGTTTTAGCTGCTCTCAACACGGTGTTTGTAGGGATGCAAACGACGTATATTTTATGGACATGGTTGGTGGAAGGACGACCACGAGCGACCATCTCGGCTTGCTTCATGTTTACTTGCCGTGGCATTCTGGGTTACTCTACTCAGCTCCCTCTTCCTCAGGATTTTCTAGGATCAGGGGTAGATTTTCCGGTAGGAAACGTCTCGTTCTT Partial coding sequence:(SEQ ID NO: 25)ATGTCAACTACAACAATCGTCCCTCTCCGTCGCACTTCTAACTCTCTCAATGAATACCACACTAACGCAGTCGCCTTTGACGGAATCGTCGGGTCAGCAAGTACTAGCCAAATGGAGGAGATTGTTACGCAAACCGACGACTGCTACGCCAACCCCAACGGAGATGGAGGGAGAAGCAAGACGTCGTTAATGACGTGGAGGATGTGCAATCCTGTCCACGTGGTGAGAGTCCATTGGATACCGTGTTTGTTTGCGGTAGGAGTTCTGTTCTTCACGTGCGTAGAGGAGTACATGCTCCAGATGATTCCGGCGAGTTCTGAGCCGTTCGATATTGGTTTTGTGGCGACGGGCTCTCTGTATCGCCTCTTGGCTTCTTCACCGGATCTTAATACCGTTTTAGCTGCTCTCAACACGGTGTTTGTAGGGATGCAAACGACGTATATTTTATGGACATGGTTGGTGGAAGGACGACCACGAGCGACCATCTCGGCTTGCTTCATGTTTACTTGCCGTGGCATTCTGGGTTACTCTACTCAGCTCCCTCTTCCTCAGGATTTTCTAGGATCAGGGGTAGATTTTCCGGTAGGAAACGTCTCGTTCTT ...Unigene Bra. 370 translated amino acid sequence) (SEQ ID NO: 26)MSTTTIVPLRRTSNSLNEYHTNAVAFDGIVGSASTSQMEEIVTQTDDCYANPNGDGGRSKTSLMTWRMCNPVHVVRVHWIPCLFAVGVLFFTCVEEYMLQMIPASSEPFDIGFVATGSLYRLLASSPDLNTVLAALNTVFVGMQTTYILWTWLVEGRPRATISACFMFTCRGILGYSTQLPLPQDFLGSGVDFPVGNVSF ...

TABLE 4 Protein identities of ROD1 and other putative homologues in B.napus, B.rapa and B. oleracea. BnROD1 Bna6194 Bra2038_2 BoES948687 ROD1ROD2 Bra370 BnROD1 100 100 96 85 76 79 Bna6194 100 96 85 76 79 Bra2038_296 85 77 80 BoES948687 74 66 74 ROD1 76 71 ROD2 68 Bra370

Example 7 Biological Materials, as Provided for Herein, that ContainRelatively High Concentrations of Long Chain Fats with ModestUnsaturation Provide Improved Feedstocks for the Production of Biodieseland Related Products

According to further aspects of the present invention, the quality of abiodiesel derives from the chemical characteristics of the constituentfats within the source biological material. While chemical and physicalprocessing can be employed to alter the fat profile during biodieselsynthesis and processing, these add cost to the end product. Thusmethods which alter the fat composition of the biological materialduring growth and maturation are particularly valuable.

Specific variables of relevance to the quality of a biodiesel derivefrom an interplay between the cloud point, oxidative stability andenergy density. For example; optimal cloud points derive from highmelting point oils, which typically are comprised of highly unsaturatedand/or short chain fats, however mixtures of this composition are oftenoxidatively unstable and have low energy densities. Similarly, optimaloxidative stability and energy density derives from oils with long chainfats with low/little unsaturation, however such mixtures typically haveundesirable low temperature cloud points.

Accordingly, biological materials, as provided for herein, that containrelatively high concentrations of long chain fats with modestunsaturation provide improved feedstocks for the production of biodieseland related products.

ADDITIONAL REFERENCES CITED IN RELATION TO EXAMPLES 3-8 (ANDINCORPORATED BY REFERENCE HEREIN, FOR THERE REFERRED TO TEACHINGS

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The invention claimed is:
 1. An oil seed-bearing plant or a partthereof, wherein the plant or part thereof is other than Arabidopsis,comprising a mutation of at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) sequence that modifies the expressionor activity thereof in one or more seeds or developing seeds of theplant, wherein the level, amount, or distribution of fatty acidunsaturation in the seed oil is modified relative to the seed oil ofplants with normal seed expression of the PDCT, and wherein the fattyacid unsaturation in seed oil is differentially reduced relative tofatty acid unsaturation in one or more membrane lipids.
 2. The oilseed-bearing plant or a part thereof of claim 1, wherein modifying theexpression or activity of the at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)comprises down-regulating at least one of the expression and activity,wherein the level, amount, or distribution of fatty acid unsaturation inthe seed oil is reduced.
 3. The oil seed-bearing plant or a part thereofof claim 1, comprising two or more different mutations that modify thelevel, amount, or distribution of fatty acid unsaturation in the seedoil, wherein at least one of the two or more different mutations is amutation of at least one phosphatidylcholine:diacylglycerolcholinephosphotransferase (PDCT) in one or more seeds or developingseeds of the plant.
 4. The oil seed-bearing plant or a part thereof ofclaim 3, wherein at least one of the two or more different mutations isa FAD2 desaturase mutation that reduces or eliminates FAD2 activity oramount in the seed or developing seed.
 5. The oil seed-bearing plant ora part thereof of claim 1, wherein the at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)comprises at least one sequence selected from the group consisting ofSEQ ID NO:3, a sequence having at least 46, at least 48%, at least 58%,at least 64%, at least 71% or at least 85% amino acid sequence identitytherewith, and PDCT-active portions thereof.
 6. The oil seed-bearingplant or a part thereof of claim 5, wherein the at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)comprises at least one sequence selected from the group consisting ofSEQ ID NOS:7, 9, 11, 13, 15, 17, 20, 22, 24, 26, and PDCT-activeportions thereof.
 7. A seed or true-breeding seed derived from the oilseed-bearing plant or a part thereof of claim 1, wherein the seedcomprises the mutation of at least onephosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)sequence that modifies the expression or activity thereof.